Treating neurological disorders using selective antagonists of persistent sodium current

ABSTRACT

The present invention provides methods of treating neurological disorders in a mammal by administering to the mammal an effective amount of a selective persistent sodium channel antagonist that has at least 20-fold selectivity for persistent sodium current relative to transient sodium current.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 10/928,949, filed Aug.27, 2004, an application that claims priority pursuant to 35 U.S.C.§119(e) to provisional application Ser. No. 60/498,902 filed Aug. 29,2003, both of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of neurobiology,physiology, biochemistry and medicine and can be directed toward thetreatment of neurological disorders and, in particular, to thetherapeutic use of compounds that selectively reduce persistent sodiumcurrents to treat neurological disorders.

2. Background Information

The lipid bilayer membrane of all cells forms a barrier that is largelyimpermeable to the flux of ions and water. Residing within the membraneare a superfamily of proteins called ion channels, which provideselective pathways for ion flux. Precisely regulated conductancesproduced by ion channels are required for intercellular signaling andneuronal excitability. Over the past 50 years, an increasing number ofdiseases of the nervous system and other excitable tissues have beenshown to result from the dysregulation of ion channels. This class ofdisease has been termed channelopathies.

In particular, a group of ion channels that open upon depolarization ofexcitable cells are classified as voltage-gated and are responsible forelectrical activity in nerve, muscle and cardiac tissue. In neurons, ioncurrents flowing through voltage-gated sodium channels are responsiblefor rapid spike-like action potentials. During action potentials themajority of sodium channels open very briefly. These brief openingsresult in transient sodium currents. However, a subset of voltage-gatedsodium channels does not close rapidly, but remain open for relativelylong intervals. These channels therefore generate sustained orpersistent sodium currents. The balance between transient and persistentsodium current is crucial for maintaining normal physiological functionand electrical signaling throughout the entire nervous system.

In conditions characterized by aberrant levels of persistent sodiumcurrent, normal function is disrupted when neurons discharge signalsinappropriately and include, e.g., neuropathies; hypoxias and ischemias;behavioral disorders and dementia; and movement and neurodegenerativediseases. For example, in the case of the neuropathies embraced byepilepsy, there can be a brief electrical “storm” arising from neuronsthat are inherently unstable because of a genetic defect as in varioustypes of inherited epilepsy, or from neurons made unstable by metabolicabnormalities such as low blood glucose, or alcohol. In other cases, theabnormal discharge can come from a localized area of the brain, such asin patients with epilepsy caused by head injury or brain tumor. In thecase of ischemic injuries, such as, e.g., cerebral ischemia andmyocardial ischemia, there can be prolonged electrical activity arisingfrom neurons in which persistent sodium channel expression or activityis increased. Such aberrant electrical activity can cause or contributeto neuronal death, which can lead to debilitating injury or death of anindividual. Aberrant electrical activity also can contribute toneurodegenerative disorders such as, without limitation, Parkinson'sdisease, Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis and multiple sclerosis.

At present, treatments for many diseases characterized by aberrantlevels of persistent sodium channel current are inadequate ornon-existent. Current therapies, such as, e.g., Berger et al., Treatmentof Neuropathic Pain, U.S. Pat. No. 5,688,830 (Nov. 18, 1997); Marquesset al., Sodium Channel Drugs and Uses, U.S. Pat. No. 6,479,498 (Nov. 12,2002); Choi et al., Sodium Channel Modulators, U.S. Pat. No. 6,646,012(Nov. 11, 2003); and Chinn et al., Sodium Channel Modulators, U.S. Pat.No. 6,756,400 (Jun. 29, 2004), encompass general sodium channelmodulators that systemically effect transient currents. As such, theusefulness of available sodium channel blocking drugs is severelylimited by potentially adverse side effects, such as, e.g., paralysisand cardiac arrest.

Thus, there exists a need to identify new therapeutic methods that canbe used to selectively treat conditions characterized by aberrant levelsof persistent sodium current, such as, e.g., neuropathies; hypoxias andischemias; behavioral disorders and dementia; and movement andneurodegenerative diseases, and to protect the brain from the damagingeffects of persistent sodium current. The present invention satisfiesthese needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods of treating a mammal having acondition characterized by aberrant levels of persistent sodium current.In one embodiment, the method involves administering to the mammal aneffective amount of a selective persistent sodium current antagonistthat has at least 20-fold selectivity for a persistent sodium currentrelative to transient sodium current. In further embodiments, theantagonist has at least 50-fold selectivity for a persistent sodiumcurrent, at least 200-fold selectivity for a persistent sodium current,at least 400-fold selectivity for a persistent sodium current, at least600-fold selectively for a persistent sodium current, or at least1000-fold selectively for a persistent sodium current, relative to atransient sodium current. A variety of mammals can be treated by themethods of the invention including, without limitation, humans.

The methods of the invention can be used to treat a variety ofconditions characterized by aberrant levels of persistent sodiumcurrent. In certain embodiments, the methods are directed to treatingneuropathies, including, without limitation, amyloidosis, autoimmunedisorders, palsies, connective tissue disorders, epilepsies, andconditions associated with neuropathies like alcoholism, cancers,infectious diseases, organ disorders and vitamin deficiencies. In otherembodiments, the methods are directed to treating hypoxic and ischemicconditions, such as, e.g., cerebral ischemia, myocardial ischemia,ischemia retinae, diabetes ischemia and postural ischemia. In stillother embodiments, the methods are directed to treating behavioraldisorders, dementia, movement disorders, and neurodegenerative diseases,such as, without limitation, Parkinson's disease, Alzheimer's disease,Huntington's disease, amyotrophic lateral sclerosis and multiplesclerosis. In further embodiments, the methods are directed to treatingdiabetic retinopathy. In yet other embodiments, the methods are directedto treating conditions characterized by aberrant levels of intracellularnitric oxide. In additional embodiments, the methods provide forreducing neuronal death associated with aberrant levels of persistentsodium current.

A variety of selective persistent sodium current antagonists can beuseful in the methods of the invention. In one embodiment, a method ofthe invention is practiced by administering an effective amount of aselective antagonist that has at least 20-fold selectivity for apersistent sodium current relative to a transient sodium current. Infurther embodiments, the antagonist has at least 50-fold selectivity fora persistent sodium current; at least 200-fold selectivity for apersistent sodium current; at least 400-fold selectivity for apersistent sodium current; at least 600-fold selectively for apersistent sodium current; or at least 1000-fold selectively for apersistent sodium current, relative to transient sodium current.

In further embodiments, the methods of the invention involveadministering an effective amount of a selective persistent sodiumcurrent antagonist belonging to one of the disclosed structural classesof selective persistent sodium current antagonists. Such a selectivepersistent sodium channel antagonist can be, without limitation, acompound represented by a formula selected from Formula 1:

wherein,

Ar¹ is an aryl group;

Ar² is an aryl group;

Y is absent or is selected from:

R¹ is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

R² and R³ are independently selected from hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy, fluoro, C₁-C₈ carbocyclic ring, or C₁-C₈heterocyclic ring;

R⁴ is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl; and

n is an integer of from 1 to 6;

Formula 4

wherein,

Ar³ is an aryl group;

Ar⁴ is an aryl group;

X¹ and Y¹ are independently selected from

R⁵ and R⁶ are independently selected from hydrogen, fluoro, C₁ to C₈alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R⁸ and R⁹ are selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl,COR¹², COCF₃;

R¹⁰ and R¹¹ are selected from hydrogen, halogen, hydroxyl, C₁-C₈ alkyl,aryl, arylalkyl, and

R¹² is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

Formula 2

wherein,

Ar⁵ is an aryl group;

Ar⁶ is an aryl group;

X² is O, S, or NR¹⁴;

Y² is N or CR¹⁵;

Z² is N or CR¹⁶;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹³ is selected from halogen, C₁-C₈ alkyl, arylalkyl, and(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁴ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁵ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁶ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂; and

c is 0 or an integer from 1 to 5; and

Formula 3

wherein,

Ar⁷ is an aryl group;

R is selected from halogen, C₁-C₈ alkyl, NR²²R²³, OR²²;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹⁷ and R¹⁸ are independently selected hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy;

R¹⁹ and R²⁰ are independently selected from hydrogen, halogen, C₁-C₈alkyl, hydroxy, amino, CF₃;

R²¹, R²², and R²³ are independently selected from hydrogen, aryl orC₁-C₈ alkyl;

a is 0 or an integer from 1 to 5; and

m is 0 or and integer from 1 to 3.

A compound corresponding to any of the above formulas also can be apharmaceutically acceptable salt, ester, amide, or geometric,steroisomer, or racemic mixture.

Any of the variety of routes of administration can be useful fortreating a neurological disorder according to a method of the invention.In particular embodiments, administration is performed peripherally,systemically or orally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows four compounds that are selective persistent sodium currentantagonists.

FIG. 2 shows inhibition of persistent current-dependent depolarizationby Na⁺ channel blockers. In the this assay, cells are resting in wellscontaining 80 μl of TEA-MeSO₃ (Na⁺-free box) to which is added 240 μl ofNaMeSO₃ buffer containing 13 mM KMeSO₃ for a final K⁺ concentration of10 mM and a final Na⁺ concentration of 110 mM (Na⁺/K⁺-addition). Thiselicits a robust depolarizing response. Following the resolution of thesodium-dependent depolarization, a second aliquot of KMeSO₃ is added tothe well bringing the final K⁺ concentration to 80 mM (Highpotassium-addition). This addition results in a second depolarizingresponse. Compounds that reduce the sodium-dependent, but not thepotassium-dependent depolarizations are selected as persistent sodiumchannel blockers. Circles indicate the control response with 0.1% DMSOadded, triangles show the effects of the sodium channel inhibitortetracaine (10 μM) and the diamonds show the response during theapplication of a non-specific channel blocker.

FIG. 3 shows data from assays in which the screening window for thepersistent current assay is determined. To evaluate the size of the“screening window,” data was examined from assays in which responses tosodium-dependent depolarization were measured in the presence of 10 μMTetracaine to completely block the sodium-dependent depolarization or inthe presence of a 0.1% DMSO control to obtain a maximum depolarization.Data were binned into histograms and a screening window (2) wascalculated from the mean and standard deviation for the maximal andminimum values according to the equation:Z=1−(3×STD_(Max)+3×STD_(Min))/(Mean_(Max)−Mean_(Min)). Histograms A, Band C represent data obtained from three different assay plates. Thescreening window for a run was considered adequate 1.0≧Z≧0.5.

FIG. 4 shows sodium current traces before and after the addition of 3 μMCompound 1 or 500 nM TTX. HEK cells expressing Na_(v)1.3 channels werepatch clamped in the perforated-patch mode. Currents were elicited by200 msec test pulses to 0 mV from a holding potential of −90 mV.

FIG. 5 shows a dose-response curve for Compound 1. The peak amplitudesof transient Na⁺ current (I_(t)) and the steady state amplitude of thepersistent current (I_(p)) were measured at various Compound 1concentrations, normalized to amplitude of the control currents. Thepercent block was then plotted against drug concentration. Solid linesrepresent fits to the data with the Hill equation. The calculated EC₅₀values and Hill coefficients are as follows: Hillslope, I_(t) is 0.354and I_(p) is 0.733; EC₅₀, I_(t) is 0.167 M and I_(p) is 3.71×10⁻⁶ M.

DETAILED DESCRIPTION OF THE INVENTION

I. Voltage-gated Sodium Channels

In the normal functioning of the nervous system, neurons are capable ofreceiving a stimulus, and in response, propagating an electrical signalaway from their neuron cell bodies (soma) along processes (axons). Fromthe axon, the signal is delivered to the synaptic terminal, where it istransferred to an adjacent neuron or other cell. Voltage-sensitivesodium channels have a critical role in nervous system function becausethey mediate propagation of electrical signals along axons.

Voltage-gated sodium channels are members of a large mammalian genefamily encoding at least 9 alpha- and 3 beta-subunits. While all membersof this family conduct Na⁺ ions through the cell membrane, they differin tissue localization, regulation and, at least in part, in kinetics ofactivation and inactivation, see, e.g., William A. Catterall, From IonicCurrents to Molecular Mechanism: The Structure and Function ofVoltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); and Sanja D.Novakovic et al., Regulation of Na ⁺ Channel Distribution in the NervousSystem, 24(8) TRENDS NEUROSCI. 473-478 (2001), which are herebyincorporated by reference in their entirety.

Generally, under resting conditions, sodium channels are closed-until astimulus depolarizes the cell to a threshold level. At this threshold,sodium channels begin to open and then rapidly generate the upstroke ofthe action potential. Normally during an action potential, sodiumchannels open briefly (one millisecond) and then close (inactivate)until the excitable cell returns to its resting potential and the sodiumchannels re-enter the resting state.

Without wishing to be bound by the following, this behavior ofvoltage-gated sodium channels can be understood as follows. Sodiumchannels can reside in three major conformations or states. The restingor “closed” state predominates at negative membrane potentials (≦−60mV). Upon depolarization, channels open and allow current to flow.Transition from the resting state to the open state occurs within amillisecond after depolarization to positive membrane potentials.Finally, during sustained depolarization (>1-2 ms), channels enter asecond closed or inactive state. Subsequent re-opening of channelsrequires recycling of channels from an inactive state to a restingstate, which occurs when the membrane potential returns to a negativevalue (repolarization). Therefore, membrane depolarization not onlycauses sodium channels to open, but also causes them to close, duringsustained depolarization.

A small fraction of the sodium channels can fail to inactivate even withsustained depolarization. This non-inactivating sodium current is calleda “persistent” sodium current. Four sodium channels, Nav1.3, Nav1.5,Nav1.6 and Nav1.9, have historically been known to generate a persistentcurrent. Recent evidence, however, suggests that all voltage-gatedsodium channels are capable of producing a persistent current, see,e.g., Abraha Taddese & Bruce P. Bean, Subthreshold Sodium Current fromRapidly Inactivating Sodium Channels Drives Spontaneous Firing ofTubermammillary Neurons, 33(4) NEURON 587-600 (2002); Michael Tri H. Do& Bruce P. Bean, Subthreshold Sodium Currents and Pacemaking ofSubthalamic Neurons: Modulation by Slow Inactivation, 39(1) NEURON109-120 (2003). The mechanism that produces a persistent current ispoorly understood. Two hypothesis are (1) that different sodium channelsproduce transient and persistent currents, and (2) that a sodium channelcapable of producing transient sodium current enters a different gatingmode to produce a persistent current. Persistent sodium channels canopen at more negative membrane potentials relative to normal sodiumchannels and inactivate at more positive potentials, see, e.g., JacopoMagistretti & Angel Alonso, Biophysical Properties and Slow-voltageDependent Inactivation of a Sustained Sodium Current in EntorhinalCortex Layer-II Principal Neurons: A Whole-Cell and Single-Channel Study114(4) J. GEN. PHYSIOL. 491-509 (1999). Persistent sodium current havebeen observed at membrane potentials as negative as −80 mV, see, e.g.,Peter K. Stys, Anoxic and Ischemic Injury of Myelinated Axons in CNSWhite Matter: From Mechanistic Concepts to Therapeutics, 18(1) J. CEREB.BLOOD FLOW METAB. 2-25 (1998) and have been shown to persist for secondsfollowing depolarization at potentials as positive as 0 mV, see, e.g.,Magistretti & Alonso, supra, (1999). Thus, persistent sodium current isdistinct from, and can be readily distinguished from, transient sodiumcurrent.

Although the physiological role of persistent sodium current is notfully understood, such current can function in generating rhythmicoscillations; integrating synaptic input; modulating the firing patternof neurons; and regulating neuronal excitability and firing frequency,see, e.g., Wayne E. Crill, Persistent Sodium Current in MammalianCentral Neurons 58 ANNU. REV. PHYSIOL. 349-362 (1996); and David S.Ragsdale & Massimo Avoli, Sodium Channels as Molecular Targets forAntiepileptic Drugs, 26(1) BRAIN RES. BRAIN RES. REV. 16-28 (1998).Aberrant persistent sodium current can contribute to the development orprogression of many pathological conditions. For example, persistentsodium current are thought to induce deleterious phenomena, including,e.g., neuropathies, cardiac arrhythmia, epileptic seizure,neurodegeneration, and neuronal cell death under hypoxic and ischemicconditions, see, e.g., Christoph Lossin et al., Molecular Basis of anInherited Epilepsy 34(6) NEURON 877-84 (2002); Peter K. Stys et al.,Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter: Role ofNa ⁺ Channels and Na ⁽⁺⁾ —Ca2⁺ Exchanger, 12(2) J. NEUROSCI. 430-439(1992); Peter K. Stys et al., Noninactivating, Tetrodotoxin-Sensitive Na⁺ Conductance in Rat Optic Nerve Axons, 90(15) PROC. NATL. ACAD. SCI.USA, 6976-6980 (1993); and Giti Garthwaite et al., Mechanisms ofIschaemic Damage to Central White Matter Axons: A QuantitativeHistological Analysis Using Rat Optic Nerve, 94(4) NEUROSCIENCE1219-1230 (1999). Thus, aberrant persistent sodium current cancontribute to development or progression of pathological conditions bycollapsing the normal cell transmembrane gradient for sodium, leading toreverse operation of the sodium-calcium exchanger, and resulting in aninflux of intracellular calcium, which injures the axon, see, e.g., Styset al., supra, (1992).

While abnormal activity of a persistent current can underlie a widearray of neurological disorders, the underlying mechanisms appears to besimilar. It is generally understood that abnormally increased persistentsodium current can depolarize the resting membrane potential or reducethe rate of repolarization during an action potential. Either effect mayproduce a state of hyper-excitability in which aberrant neuronalbehavior is manifested. This aberrant neuronal behavior can result in aneuron with increased firing rates, enhanced sensitivity to synapticinput or abnormal repetitive or rhythmic firing patterns. It is alsogenerally understood that abnormally high levels of persistent currentgenerate sustained membrane depolarization and a concomitant increase ofNa⁺ within the cell. This high Na⁺ influx, in turn, drives thesodium/calcium exchanger, which in turn, results in detrimental levelsof Ca²⁺ to accumulate inside affected cells. Abnormally high levels ofCa²⁺ result in neuronal cell dysfunction and neuronal cell death. Thus,by collapsing the normal cell transmembrane gradient for sodium, apersistent current can reverse the operation of the sodium-calciumexchanger, and the resulting an influx of intracellular calcium wouldcause injures or death to a nerve. As disclosed herein, conditionsassociated with aberrant persistent sodium current can be treated byselectively inhibiting or reducing persistent sodium current withoutcompromising normal transient sodium current function, thereby allowingnormal neuronal function (excitability).

II. Neurological Disorders and Persistent Sodium Current

The methods of the invention can be used to reduce or eliminate aberrantlevels of persistent sodium current in a mammal, and thus can be used,for example, to treat any of a variety of neurological conditions thatinvolve aberrant levels of persistent sodium current. Neuronaldisturbance, including neuronal dysfunction and neuronal death,associated with unwanted persistent neuronal firing can contribute to,or cause, a variety of disorders of the central and peripheral nervoussystems. Therefore, a compound that decreases persistent sodium currentwithout a similar decrease in non-pathological transient sodium currentcan effectively treat such conditions, without harmful side effects thatgenerally accompany non-selective sodium channel blockers currently inuse. Because all sodium channels seem capable of generating a persistentcurrent, and since any condition whose underlying cause includes anaberrant persistent sodium current, a very wide range of neurologicalabnormalities can be treated using a persistent sodium channelantagonist. Conditions that can be treated according to a method of theinvention include, without limitation, neuropathies such as, e.g.,epilepsies, palsies, connective tissue disorders and conditionsassociated with neuropathies, like, alcoholism, cancers, infectiousdiseases, organ disorders and vitamin deficiencies; hypoxia andischemia, such as, e.g., cerebral hypoxia/ischemia, myocardialhypoxia/ischemia, myoischemia, diabetes ischemia and hypoxia/ischemicretinopathy; and behavioral disorders, dementia, movement disorders andneurodegenerative conditions such as. e.g., Parkinson's disease,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosisand multiple sclerosis.

Based on the identification of selective persistent sodium currentantagonists that have at least 20-fold selectivity for persistent sodiumcurrent relative to transient sodium current, the present inventionprovides therapeutic methods that involve selectively antagonizingpersistent sodium channel. Whereas certain conditions have been treatedusing non-selective sodium channel blockers, albeit with significantside effects, the methods of the present invention involve administeringto a mammal an effective amount of a selective persistent sodium currentantagonist that has at least 20-fold selectivity for persistent sodiumcurrent relative to transient sodium current.

By preventing or reducing aberrant levels of persistent sodium current,the progression of various conditions associated with unwantedpersistent neuronal firing can be stopped or slowed, and improvement inthe pathophysiology or symptoms appreciated. As used herein, the term“conditions associated with unwanted persistent neuronal firing” means adisorder in which persistent membrane sodium conductance causes orcontributes to functional changes resulting from disease or injury. Suchfunctional changes, or pathophysiology, can involve either neuronaldamage, including neuronal death; unwanted persistent neuronal firing;or both. As used-herein, the term “reducing,” when used in reference toneuronal death means preventing, decreasing or eliminating unwantedpersistent neuronal firing or aberrant levels of persistent sodiumcurrent. Reducing aberrant levels of persistent sodium current byadministering a selective persistent sodium current antagonist can be aneffective method for treating conditions involving neuronal dysfunctionor neuronal death, for example, for treating conditions characterized byaberrant levels of persistent sodium current or aberrant levels ofintracellular nitric oxide.

III. Treatment of Neuropathies Using a Selective Persistent SodiumCurrent Antagonist

The present invention provides methods of treating a neuropathy byadministering an effective amount of a selective persistent sodiumcurrent antagonist having at least 20-fold selectivity for persistentsodium current relative to transient sodium current. Aberrant levels ofsodium current are associated with a variety of neuropathic conditionsthat led to neuronal dysfunction or neuronal death. As used herein, theterm “neuropathic condition” means any condition resulting in nervedamage, including, e.g., motor nerve damage, sensory nerve damage,autonomic nerve damage. Neuropathic conditions include a heterogeneousgroup of conditions of the central or peripheral nervous system thatinclude, without limitation, headache, pain, inflammatory diseases,movement disorders, tumors, birth injuries, developmental abnormalities,neurocutaneous disorders, autonomic disorders, and paroxysmal disorders.As such, a neuropathic condition have a wide range of differentetiologies, including, e.g., hereditary or sporatic, secondary to atoxic or metabolic process, and can result from an injury, trauma,disease, or infection. Such conditions can be characterized byabnormalities of relatively specific regions of the brain or specificpopulations of neurons. The particular cell groups affected in differentneuropathic conditions typically determine the clinical phenotype of thecondition.

Exemplary examples of neuropathies include, without limitation,amyloidosis; autoimmune disorders such as, e.g., Guillain-Barrésyndrome, Hashimoto's thyroiditis, pernicious anemia, Addison's disease,type I diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease;movement disorders like palsies involving injury or damage to nervessuch as, e.g., cerebral palsy, Bell's palsy, diver's palsy,extrapyramidial cerebral palsy, lead palsy, Ramsey Hunt syndrome,obstetrical palsy, like Erb palsy and Klumpke palsy, posticus palsy,Scrivener's palsy, tardy median palsy, tardy ulnar palsy, andprogressive supranuclear palsy; arthritis/connective tissue disorderssuch as, e.g., osteoarthritis, rheumatoid arthritis, juvenile arthritis,gouty arthritis; spondyloarthritis, scleroderma, fibromyalgia,osteoporosis, noise sensitivity, multiple chemical sensitivity andasthma; conditions associated with neuropathies like alcoholism,cancers, infectious diseases, organ disorders and vitamin deficiencies;and epilepsies, seizures and paroxysmal conditions. The skilled personunderstands that these and other mild, moderate or severe neuropathicconditions can be treated according to a method of the invention.

As a non-limiting example, epilepsies are conditions that can becharacterized by aberrant levels of persistent sodium current.Epilepsies and other seizure disorders, are a group of neuronaldysfunction disorders of the central nervous system and are generallycharacterized by sudden seizures, muscle contractions, and partial ortotal loss of consciousness. Epilepsy is a disorder characterized by theoccurrence of at least 2 unprovoked seizures. Seizures are themanifestation of abnormal hypersynchronous discharges of corticalneurons. The clinical signs or symptoms of seizures depend upon thelocation and extent of the propagation of the discharging corticalneurons. That seizures are a common nonspecific manifestation ofneurologic injury and disease should not be surprising, because the mainfunction of the brain is the transmission of electrical impulses. Thelifetime likelihood of experiencing at least one epileptic seizure isabout 9%, and the lifetime likelihood of being diagnosed as havingepilepsy is almost 3%. However, the prevalence of active epilepsy isonly 0.8%.

Epilepsies can be divided into two major categories. Partial-onsetseizures begin in one focal area of the cerebral cortex, whilegeneralized-onset seizures have an onset recorded simultaneously in bothcerebral hemispheres. Some seizures are difficult to fit into oneparticular class, and they are considered as unclassified seizures.Partial-onset seizures include, e.g., simple partial seizures, complexpartial seizures and secondarily generalized tonic-clonic seizures.Generalized-onset seizures include, e.g., absence seizures, tonicseizures, clonic seizures, myoclonic seizures, primary generalizedtonic-clonic seizures, and atonic seizures. Likewise, epilepticsyndromes can be classified into two major groups, localization-relatedsyndromes and generalized-onset syndromes.

Voltage-gated sodium channels, which play an important role ininitiation and transmission of action potentials, are involved in theetiology of epilepsy, and appear to include Na_(v)1.1, Na_(v) 1.2,Na_(v) 1.3, Na_(v) 1.5, and Na_(v) 1.6, see, e.g., Rudiger Kohling,Voltage-gated Sodium Channels in Epilepsy, 43(11) Epilepsia 1278-1295(2002); Michael M. Segal Sodium Channels and Epilepsy Electrophysiology,241 NOVARTIS FOUND. SYMP. 173-180 (2002), which are hereby incorporatedby reference in their entirety. Examination of individuals sufferingfrom hereditary forms of epilepsy has revealed these individuals carrieddeleterious mutations in Na_(v)1.1 or Na_(v)1.2, see, e.g., Lossin etal., supra, (2002); Miriam H. Meisler et al., Mutations of Voltage-gatedSodium Channels in Movement Disorders and Epilepsy, 241 NOVARTIS FOUND.SYMP. 72-81 (2002); J. Spampanato et al., Generalized Epilepsy withFebrile Seizures Plus Type 2 Mutation W1204R Alters Voltage-DependentGating of Na(V) 1.1 Sodium Channels, 116(1) NEUROSCIENCE 37-48 (2003);Paolo Bonanni et al., Generalized Epilepsy with Febrile Seizures Plus(GEFS+): Clinical Spectrum in Seven Italian Families Unrelated to SCN1A,SCN1B, And GABRG2 Gene Mutations, 45(2) EPILEPSIA 149-158 (2004); BertenP. G. M. Ceulemans et al., Clinical Correlations Of Mutations in theSCN1A Gene: from Febrile Seizures to Severe Myoclonic Epilepsy inInfancy, 30(4) PEDIATR. NEUROL. 236-243 (2004); Goryu Fukuma et al.,Mutations of Neuronal Voltage-Gated Na ⁺ Channel Alpha 1 Subunit GeneSCN1A in Core Severe Myoclonic Epilepsy in Infancy (SMEI) and inBorderline SMEI (SMEB), 45(2) EPILEPSIA 140-148 (2004); and KazusakuKamiya et al., A Nonsense Mutation of the Sodium Channel Gene SCN2A in aPatient with Intractable Epilepsy and Mental Decline, 24(11) J.NEUROSCI. 2690-2698 (2004), which are hereby incorporated by referencein their entirety. In addition, epilepsy appears to be caused byabnormal nerve discharges in the brain that result in aberrant levels ofpersistent sodium current, see, e.g., Newton Agrawal et al., IncreasedPersistent Sodium Currents in Rat Entorhinal Cortex Layer V Neurons in aPost-Status Epilepticus Model of Temporal Lobe Epilepsy, 44(12)EPILEPSIA 1601-1604 (2003); and Martin Vreugdenhil et al., PersistentSodium Current in Subicular Neurons Isolated from Patients with TemporalLobe Epilepsy, 19(10) EUR. J. NEUROSCI. 2769-2778 (2004), which arehereby incorporated by reference in their entirety. Both Na_(v)1.1 andNa_(v)1.6 are thought to be capable of producing a persistent current,see, e.g., Joshua P. Klein et al., Dysregulation of Sodium ChannelExpression in Cortical Neurons in a Rodent Model of Absence Epilepsy,1000(1-2) BRAIN RES. 102-109 (2004), which is hereby incorporated byreference in its entirety. In view of the role of persistent sodiumcurrents in epilepsy, a selective persistent sodium current antagonistcan be advantageously used to treat epilepsy without deleterious sideeffects associated with non-selective sodium channel blockers.

IV. Treatment of Hypoxias and Ischemias Using a Selective PersistentSodium Current Antagonist

The present invention also provides methods of treating a hypoxia orischemia by administering an effective amount of a selective persistentsodium current antagonist having at least 20-fold selectivity forpersistent sodium current relative to transient sodium current. Neuronaldamage or death occurring as a result of changes induced by hypoxia orischemia appears to be associated with increased persistent sodiumcurrent, see, e.g., Anna K. M. Hammarström & Peter W. Gage, Hypoxia andPersistent Current, 31 (3) EUR. BIOPHYS. J. 323-330 (2002), which ishereby incorporated by reference in its entirety. As used herein, theterm “hypoxia” means an incident during which the oxygen supply to atissue is diminished or eliminated. A hypoxia can include, e.g.,cerebral hypoxia, diffusion hypoxia, hypoxic hypoxia, cell hypoxia,ischemic hypoxia, or any other accidental or purposeful reduction orelimination of oxygen supply to a tissue. As used herein, the term“ischemia” means an incident during which the blood supply to a tissueis reduced or completely obstructed. An ischemia can include, e.g.,cerebral ischemia, myocardiac ischemia, myoischemia, diabetes ischemia,ischemia retinae, postural ischemia, or any other accidental orpurposeful reduction or complete obstruction of blood supply to atissue. That a reduction or complete obstruction of blood to a tissuenecessarily means a reduction or elimination of oxygen supply to thattissue, ischemias and hypoxias are usually related. The skilled personunderstands that these and other mild, moderate or severe hypoxic andischemic conditions can be treated according to a method of theinvention.

As a non-limiting example, cerebral ischemia occurs when a blood vesselbringing oxygen and nutrients to the brain bursts or is clogged by ablood clot or other material. Because of this rupture or blockage, partof the brain is deprived of its normal blood flow and the oxygen itcontains. In the absence of oxygen, nerve cells in the affected area ofthe brain undergo deleterious changes and die. This neuronal cell deathcan lead to a stroke, resulting in loss of control of the body partnormally controlled by these nerve cells. The devastating effects ofstroke are often permanent because damaged nerve cells are not replaced.

Cerebral hypoxia or ischemia can result, without limitation, from CNSsurgery, open heart surgery or any procedure during which the functionof the cardiovascular system is compromised; trauma that results inreduction of blood flow to the brain; disease that causes reduction ofblood flow to the brain, including cerebrovascular disease, such aschronic subdural hematoma, cavernous angioma, arteriovenousmalformation, vascular dementia, carotid or circle of Willishypertensive encephalopathy, multiple embolic infarctions, hypertensiveencephalopathy and cerebral hemorrhage; infectious diseases that cancause cranial swelling that reduces blood flow to neurons, such asmeningitis, Lyme encephalopathy, Herpes encephalitis, Creutzfeld-Jakobdisease, cerebral toxoplasmosis and the like; trauma, such as headtrauma and traumatic brain injury that cause a reduction in blood flowto neurons; and proliferative disorders that cause a reduction in bloodflow to neurons, including diseases associated with the overgrowth ofconnective tissues, such as various fibrotic diseases, vascularproliferative disorders, and benign tumors. Proliferative disorders ofthe central nervous system include, for example, cerebellarastrocytomas, and medulloblastomas, ependymomas, gliomas, germinomas,and metastatic adenocarcinoma, metastatic bronchogenic carcinoma,meningioma, sarcoma and neuroblastoma.

Sodium channel inhibitors, such as, tetrotoxin (TTX) and lidocaine, andextracelluar Na⁺ ions protect neurons from hypoxic and ischemic damage,suggesting that voltage-gated sodium channel activity is an early andimportant step in sensing oxygen levels and cell damage in neurons. Itwas subsequently shown that these oxygen sensing channels generated apersistent current, and hypoxic/ischemic conditions increased theactivity of these persistent current channels that result in anabnormally high intake of Na⁺., see, e.g., Anna H. K. Hammarström &Peter W. Gage, Oxygen-sensing Persistent Sodium Channels in RatHippocampus, 529(1) J. PHYSIOL. 107-118 (2000), which is herebyincorporated by reference in its entirety. The influx of Na⁺ would drivethe sodium/calcium exchanger, which in turn, would result in detrimentallevels of Ca²⁺ accumulate inside affected cells and cell death, see,e.g., Peter Lipton, Ischemic Cell Death in Brain Neurons, 79(4) PHYSIOL.REV. 1431-1568 (1999), which is hereby incorporated by reference in itsentirety. Therefore, application of a selective persistent sodiumcurrent antagonist can serve as a neuroprotectant against cerebralhypoxia or ischemia, without the deleterious side effects associatedwith non-selective sodium channel blockers.

As another non-limiting example, myocardial ischemia is a disorder ofcardiac function caused by insufficient blood flow to the muscle tissueof the heart. The decreased blood flow may be due to narrowing of thecoronary arteries (coronary arteriosclerosis), to obstruction by athrombus (coronary thrombosis), or less commonly, to diffuse narrowingof arterioles and other small vessels within the heart. Severeinterruption of the blood supply to the myocardial tissue results in aconcomitant interruption in oxygen which may lead to necrosis of cardiacmuscle (myocardial infarction).

Abnormal levels of a persistent sodium current, which become prominentfollowing cardiac hypoxia or ischemia, are associated with arrhythmias,which can trigger a heart attack, see, e.g., Hammarström & Gage, supra,(2002). Cardiac cells, such as, e.g., Purkinje fibers and ventricularmyocytes, generate a persistent sodium current. Examination ofventricular myocytes in the presence or absence of oxygen indicates thatpersistent sodium current increases during hypoxia, and that thisaberrant current could trigger early after depolarization, arrhythmia,and heart failure, see, e.g., Yue-Kun Ju et al., Hypoxia IncreasesPersistent Sodium Current in Rat Ventricular Myocytes, 497(2) J.PHYSIOL. 337-347 (1996), which is hereby incorporated by reference inits entirety. Additionally, application of tetrotoxin (TTX), avoltage-gated sodium channel inhibitor, reduces the action potentialduration of a persistent current in human ventricular myocytes, as wellas abolishes the early after depolarization in myocytes isolated fromheart failure patients, see, e.g., Victor A. Maltsev et al., Novel,Ultraslow Inactivating Sodium Current in Human VentricularCardiomyocytes, 98(23) CIRCULATION 2545-2552 (1998), which is herebyincorporated by reference in its entirety.

Several voltage-gated sodium channels are localized in specific regionsof the heart where they are believed to regulate distinct activities.The persistent sodium channel Na_(v)1.5 is found in the myocardium andintercalated disks/AV node and seems to be involved primarily ininitiation and propagation of the action potential from cell to cell. Onthe other hand, Na_(v)1.1 and Na_(v)1.3 appear to generate a persistentcurrent in the transverse tubules/SA node and may function incoordinating and synchronizing the action potential from the cellsurface into the interior, see, e.g., Sebastian K. G. Maier et al., AnUnexpected Requirement for Brain-Type Sodium Channels for Control ofHeart Rate in the Mouse Sinoatrial Node, 100(6) PROC. NATL. ACAD. SCI.U.S.A. 3507-3512 (2003); and Sebastian K. G. Maier et al., DistinctSubcellular Localization of Different Sodium Channel Alpha and BetaSubunits in Single Ventricular Myocytes from Mouse Heart, 109(11)CIRCULATION 1421-1427 (2004), which are hereby incorporated by referencein their entirety. Furthermore, a missense mutation in Na_(v)1.5 thataccelerates channel activation is associated with individuals diagnosedwith cardiac arrhythmia, see, e.g., Igor Splawski et al., Variant ofSCN5A Sodium Channel Implicated in Risk of Cardiac Arrhythmia, 297SCIENCE 1333-1336 (2002), which is hereby incorporated by reference inits entirety. Thus, as seen in cerebral hypoxia/ischemia, as describedabove, elevated Na⁺ levels due to an increased persistent current, willcause the sodium/calcium exchanger to import abnormally high levelsCa²⁺, thereby triggering myocardial cell death. Thus, a selectivepersistent sodium current antagonist can be used beneficially to preventcardiac hypoxia or ischemia without the harmful side effects associatedwith current non-selective sodium channel blockers.

In a third non-limiting example, ischemia retinae is a diminished bloodsupply in the retina due to diminished or failed blood circulation thatcan result in bilateral transitory or permanent blindness. Ischemia ofthe neuroretina and optic nerve can arise during an embolism, such as,e.g., retinal branch vein occlusion, retinal branch artery occlusion,central retinal artery occlusion, central retinal vein occlusion; as aresult of a disease, such, e.g., diabetic retinopathy; duringintravitreal surgery; by poisoning, such as, e.g., quinine; in retinaldegenerations such as, e.g., retinitis pigmentosa, and in age-relatedmacular degeneration; during an inflammation; during an infection; orexsanguination from recurring profuse haemorrhages (e.g., inparturition, gastric and duodenal ulcers, and pulmonary tuberculosis).The skilled person understands that the methods of the invention can beused to treat these and other types of ischemia known in the art.

The earliest ophthalmolscopic indication of an ischemic retinopathy isthe appearance of microaneruysms, which correspond with areas of focalischemia, see, e.g., Thomas W. Gardner et al., Diabetic Retinopathy:More than Meets the Eye, 47(Suppl. 2) SURV. OPHTHALMOL. S253-S262(2002); and Alistair J. Barder, A New View of Diabetic Retinopathy: ANeurodegenerative Disease of the Eye, 27(2) PROG. NEUROPSYCHOPHARMACOL.BIOL. PSYCHIATRY. 283-290 (2003), which are hereby incorporated byreference in their entirety. Coincident with or preceding these clinicalfindings, significant electrophysiological changes can be observed,including reduction in oscillatory potentials, delays in visual evokedpotentials and changes in pattern and multi-focal electroretinograms,see, e.g., Erich Lieth et al., Retinal Neurodegeneration: EarlyPathology in Diabetes, 28(1) CLIN. EXPERIMENT. OPHTHALMOL. 3-8 (2000),which is hereby incorporated by reference in its entirety. Alterationsin the normal ionic conductances of the neural retinia, includingretinal ganglion cells and their axons, have been associated withischemic retinopathy, see, e.g., Stefan Quasthoff, The Role of AxonalIon Conductances in Diabetic Neuropathy: A Review, 21(10) MUSCLE NERVE1246-1255 (1998), which is hereby incorporated by reference in itsentirety. Key observations include a decrease in conduction velocity, adysfunction of nodal sodium channels and an increase in intracellularNa⁺ concentration, see, e.g., Tom Brismar, Abnormal Na-Currents inDiabetic Rat Nerve Nodal Membrane, 10(Suppl. 2) DIABET. MED. 110S-112S(1993), which is hereby incorporated by reference in its entirety. Theincreased influx of Na⁺ would drive the sodium/calcium exchanger toimport abnormally high levels of intracellular calcium, a major cause ofneuronal cell death, see, e.g., Lipton, supra, (1999). The gradual lossof neurons in the retina indicates that progress of the disease isultimately irreversible, since these cells cannot usually be replaced.Selectively reducing persistent sodium current can provide an effectivemeans for reducing symptoms or pathophysiology of retinal ischemia.Thus, analogous to the neuroprotective effects of selective persistentsodium current blockers during cerebral hypoxia/ischemia, the presentinvention discloses a method that prevents retinal ischemias.

V. Treatment of Neurodegenerative Conditions Using a SelectivePersistent Sodium Current Antagonist

The present invention further provides methods of treating aneurodegenerative condition by administering an effective amount of aselective persistent sodium current antagonist having at least 20-foldselectivity for persistent sodium current relative to transient sodiumcurrent. Aberrant levels of sodium current are associated with a varietyof neurodegenerative conditions. As used herein, the term“neurodegenerative condition or disorder” means a conditioncharacterized by progressive loss of neural tissue. Neurodegenerativeconditions include a heterogeneous group of aberrant conditions of thecentral or peripheral nervous system that include, without limitation,behavioral disorders, dementia, neuromuscular disorders, movementdisorders, inflammatory disorders and demyelinating diseases. Suchconditions have many different etiologies such as, without limitation,sporatic or hereditary, secondary to toxic or metabolic processes, andcan result from an injury, a trauma, a disease, or an infection.Neurodegenerative conditions are progressive conditions that can be ageassociated or chronic. Such conditions can be characterized byabnormalities of relatively specific regions of the brain or specificpopulations of neurons. The particular cell groups affected in differentneurodegenerative conditions typically determine the clinical phenotypeof the condition. In particular, neurodegenerative conditions can beassociated with atrophy of a particular affected central or peripheralnervous system structure, and aberrant levels of sodium current andsubsequent elevation of intracellular sodium can be a cause orcontributing factor to this atrophy.

Exemplary neurodegenerative conditions include, but are not limited to,Motor Neuron Disease (ALS), Parkinsonian Syndromes, diffuse sclerosis,amyotrophic lateral sclerosis, multiple sclerosis, diffuse cerebralcortical atrophy, Lewy-body dementia, Pick disease, mesolimbocorticaldementia, thalamic degeneration, bulbar palsy, Huntington chorea,cortical-striatal-spinal degeneration, cortical-basal ganglionicdegeneration, cerebrocerebellar degeneration, familial dementia withspastic paraparesis, polyglucosan body disease, Shy-Drager syndrome,olivopontocerebellar atrophy, progressive supranuclear palsy, dystoniamusculorum deformans, Hallervorden-Spatz disease, Meige syndrome,familial tremors, Gilles de la Tourette syndrome, acanthocytic chorea,Friedreich ataxia, Holmes familial cortical cerebellar atrophy, AIDSrelated dementia, Gerstmann-Straussler-Scheinker disease, progressivespinal muscular atrophy, progressive balbar palsy, primary lateralsclerosis, hereditary muscular atrophy, spastic paraplegia, peronealmuscular atrophy, hypertrophic interstitial polyneuropathy, heredopathiaatactica polyneuritiformis, optic neuropathy, diabetic retinopathy,Alzheimer's disease and ophthalmoplegia. The skilled person understandsthat these and other mild, moderate or severe neurodegenerativeconditions can be treated according to a method of the invention.

As a non-limiting example, multiple sclerosis is a condition that can becharacterized by aberrant levels of persistent sodium current. MultipleSclerosis (MS) an chronic inflammatory disease of the central nervoussystem affecting white matter tissue impacts more than 350,000 personsin the United States and world-wide may affect as many as 30 cases per100,000 population. MS can therefore be considered a nerve fiber, oraxonal disease. MS can cause damage in a random manner within the CNScausing lesions or plaques to appear in CNS axons. A lesion ischaracterized by a loss of myelin (demyelination), the material thatinsulates axons. Demyelination profoundly effects the electricalproperties of the axon, slowing or blocking nervous impulses fromoccurring. A variety of bodily functions are affected as a result of theadverse effects on axon physiology. During the course of the diseaseaxons are destroyed classifying MS as a neurodegenerative disease. Manypeople with the disorder are affected during what normally would be themost productive years of their lives since the age of onset is oftenbetween 28 and 35. Drug therapies currently available at best may slowdown the disease or lessen the symptoms. It is obvious that there is anunmet need for therapies to treat this form of neurological disorder.

This neurodegenerative condition is typically marked by lack of musclecoordination, muscle weakness, speech problems, paresthesia, and visualimpairments. In human patients with multiple sclerosis as well as animalmodels of this condition, there is evidence that onset of multiplesclerosis produces changes in the expression pattern of sodium channelswithin Purkinje cells. Dysregulated sodium channel expression cancontribute to symptoms of multiple sclerosis. For example, a persistentsodium current can trigger calcium-mediated axonal injury via reversesodium-calcium exchange, see, e.g., Stephen G. Waxman, Sodium Channelsas Molecular Targets in Multiple Sclerosis, 39(2) J. REHABIL. RES. D EV.233-242 (2002); and Stephen G. Waxman, Ion Channels and NeuronalDysfunction in Multiple Sclerosis, 59(9) ARCH. NEUROL. 1377-1380 (2002),which are hereby incorporated by reference in their entirety. Inmyelinated axons, voltage-gated sodium channels Na_(v)1.2 and Na_(v)1.6specifically cluster at the nodes of Ranvier. However, both exhibitaltered expression along demyelinated axons derived from patientssuffering with multiple sclerosis. In addition, Na_(v)1.6 and thesodium/calcium exchanger co-localize within axons expressing β-APP, amaker of axonal injury in multiple sclerosis. Thus in patients sufferingfrom multiple sclerosis, altered distribution of Na_(v)1.6 is thought toproduce a persistent current that results in aberrantly high influx ofNa⁺ which drives a sodium/calcium exchanger to import abnormally highlevels of intraaxonal calcium, which triggers the neuronal damage seenin these individuals, see, e.g., Matthew J. Craner et al., MolecularChanges in Neurons In Multiple Sclerosis: Altered Axonal Expression ofNa _(v)1.2 and Na _(v)1.6 Sodium Channels And Na ⁺ /Ca2+ Exchanger,101(21), PROC. NATL. ACAD. SCI. U.S.A. 8168-8173 (2004); and Matthew J.Craner et al., Co-Localization of Sodium Channel Na _(v)1.6 and theSodium-Calcium Exchanger at Sites of Axonal Injury in the Spinal Cord inEAE, 127(2) BRAIN 294-303 (2004), which are hereby incorporated byreference in their entirety. Thus, selectively reducing this abnormallyhigh persistent sodium current can provide an effective means fortreating an individual having multiple sclerosis.

As another non-limiting example, amyotrophic lateral sclerosis (ALS) or“Motor Neuron Disease” is a neurodegenerative disorder of both the upperand lower motor neurons. The mean age of onset is approximately 55 yearsand the incidence of ALS is about two per 100,000. The prevalence of ALSin the USA is about 11 per 100,000 affecting approximately 30,000people. There are about 5,000 new cases per year, or 15 per day. ALS ischaracterized by progressive weakness of the lower and upper extremitiesas well as stiffness, muscle twitching and shaking and muscle atrophy.ALS is a fatal disease with only 20% of those inflicted surviving 5years. At present Riluzole is only FDA-approved drug that appears toslow down progression of the disease.

The etiology of ALS is unknown but one hypothesis proposes thatglutamate excitotoxicity causes neuronal cell death associated with thedisease. Interestingly, in an in vitro model of neuronal excitotoxicity,voltage-gated sodium channels, NMDA receptors and glutamate release wereshown to mediate delayed neurodegeneration via nitric oxide formation,see, e.g., Paul J. Strijbos et al, Vicious Cycle Involving Na ⁺Channels, Glutamate Release, and NMDA Receptors Mediates DelayedNeurodegeneration Through Nitric Oxide Formation, 16(16) J. NEUROSCI.5004-5013 (1996), which is hereby incorporated by reference in itsentirety. It is thought that glutamate release requires the activationof voltage-gated Na⁺ channels and therefore blocking these channels canprevent cytotoxic effects from excess spillover of glutamate. In atransgenic mouse model of ALS it was found that the morphologicalchanges associated with neurogenenration in the peripheral axons ofthese mice were accompanied by changes in membrane conductance andexcitability Jasna Kriz et al, Altered Ionic Conductances in Axons OfTransgenic Mouse Expressing the Human Neurofilament Heavy Gene: A MouseModel of Amyotrophic Lateral Sclerosis, 163(2) EXP. NEUROL. 414-421(2000). These authors suggested that the inactivation rate of the sodiumchannels from the axons of the transgenic mice were significantly slowedcompared to controls. Moreover, of the many drugs tested for ALS theonly drug shown to slow the progression of the disease (Riluzole) wasfound to block voltage-gated Na⁺ channels and subsequent glutamaterelease, see, e.g., Alessandro Stefani et al., Differential Inhibitionby Riluzole, Lamotrigine, and Phenytoin of Sodium and Calcium Currentsin Cortical Neurons: Implications for Neuroprotective Strategies, 147(1)EXP. NEUROL. 115-122 (1997); and Thomas Anger et al., MedicinalChemistry of Neuronal Voltage-Gated Sodium Channel Blockers, 44(2) J.MED. CHEM. 115-137 (2001), which are hereby incorporated by reference intheir entirety. It was subsequently shown that Riluzole targetspersistent sodium currents, see, e.g., Andrea Urbani & OttorinoBelluzzi, Riluzole Inhibits the Persistent Sodium Current in MammalianCNS Neurons, 12(10) EUR. J. NEUROSCI. 3567-3574 (2000); and FrancescaSpadoni et al., Lamotrigine Derivatives and Riluzole Inhibit INa,P inCortical Neurons, 13(9) NEUROREPORT. 1167-1170 (2002), which are herebyincorporated by reference in their entirety. Thus persistent sodiumcurrents appear to play a role in the progression of ALS, andselectively reducing this aberrantly high persistent sodium current canprovide an effective means for treating an individual having ALS.

VI. Treatment of Ocular Conditions Using a Selective Persistent SodiumCurrent Antagonist

The present invention also provides a method for treating an ocularcondition by administering an effective amount of a selective persistentsodium current antagonist having at least 20-fold selectivity forpersistent sodium current relative to transient sodium current. Unwantedneuronal firing and neuronal death induced by aberrant levels ofpersistent sodium channels can be a cause or contributing factor inocular conditions.

An ocular condition can include a disease, aliment or condition whichaffects or involves the eye or one of the parts or regions of the eye.Broadly speaking the eye includes the eyeball and the tissues and fluidswhich constitute the eyeball, the periocular muscles (such as theoblique and rectus muscles) and the portion of the optic nerve which iswithin or adjacent to the eyeball. An anterior ocular condition is adisease, ailment or condition which affects or which involves ananterior (i.e. front of the eye) ocular region or site, such as aperiocular muscle, an eye lid or an eye ball tissue or fluid which islocated anterior to the posterior wall of the lens capsule or ciliarymuscles. Thus, an anterior ocular condition primarily affects orinvolves, the conjunctiva, the cornea, the conjunctiva, the anteriorchamber, the iris, the posterior chamber (behind the retina but in frontof the posterior wall of the lens capsule), the lens or the lens capsuleand blood vessels and nerve which vascularize or innervate an anteriorocular region or site. A posterior ocular condition is a disease,ailment or condition which primarily affects or involves a posteriorocular region or site such as choroid or sclera (in a position posteriorto a plane through the posterior wall of the lens capsule), vitreous,vitreous chamber, retina, optic nerve (i.e. the optic disc), and bloodvessels and nerves which vascularize or innervate a posterior ocularregion or site.

Thus, a posterior ocular condition can include a disease, ailment orcondition, such as for example, macular degeneration (such asnon-exudative age related macular degeneration and exudative age relatedmacular degeneration); choroidal neovascularization; acute macularneuroretinopathy; macular edema (such as cystoid macular edema anddiabetic macular edema); Behcet's disease, retinal disorders, diabeticretinopathy (including proliferative diabetic retinopathy); retinalarterial occlusive disease; central retinal vein occlusion; uveiticretinal disease; retinal detachment; ocular trauma which affects aposterior ocular site or location; a posterior ocular condition causedby or influenced by an ocular laser treatment; posterior ocularconditions caused by or influenced by a photodynamic therapy;photocoagulation; radiation retinopathy; epiretinal membrane disorders;branch retinal vein occlusion; anterior ischemic optic neuropathy;non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa andglaucoma. Glaucoma can be considered a posterior ocular conditionbecause the therapeutic goal is to prevent the loss of or reduce theoccurrence of loss of vision due to damage to or loss of retinal cellsor optic nerve cells (i.e. neuroprotection).

An anterior ocular condition can include a disease, ailment orcondition, such as for example, aphakia; pseudophakia; astigmatism;blepharospasm; cataract; conjunctival diseases; conjunctivitis; cornealdiseases; corneal ulcer; dry eye syndromes; eyelid diseases; lacrimalapparatus diseases; lacrimal duct obstruction; myopia; presbyopia; pupildisorders; refractive disorders and strabismus. Glaucoma can also beconsidered to be an anterior ocular condition because a clinical goal ofglaucoma treatment can be to reduce a hypertension of aqueous fluid inthe anterior chamber of the eye (i.e. reduce intraocular pressure).

Examples of ocular conditions that can be treated using a method of theinvention include, but are not limited to, maculopathies and retinaldegeneration such as, e.g., Non-Exudative Age Related MacularDegeneration (ARMD), Exudative Age Related Macular Degeneration (ARMD),Choroidal Neovascularization, Diabetic Retinopathy, Central SerousChorioretinopathy, Cystoid Macular Edema, Diabetic Macular Edema, MyopicRetinal Degeneration; retinal inflammatory diseases, such as, e.g.,Acute Multifocal Placoid Pigment Epitheliopathy, Behcet's Disease,Birdshot Retinochoroidopathy, Infectious (Syphilis, Lyme, Tuberculosis,Toxoplasmosis), Intermediate Uveitis (Pars Planitis), MultifocalChoroiditis, Multiple Evanescent White Dot Syndrome (MEWDS), OcularSarcoidosis, Posterior Scleritis, Serpiginous Choroiditis, SubretinalFibrosis and Uveitis Syndrome, Vogt-Koyanagi-Harada Syndrome, PunctateInner Choroidopathy, Acute Posterior Multifocal Placoid PigmentEpitheliopathy, Acute Retinal Pigement Epitheliitis, Acute MacularNeuroretinopathy; retinal vascular and exudative diseases, such as,e.g., Diabetic retinopathy, Central Retinal Arterial Occlusive Disease,Central Retinal Vein Occlusion, Disseminated Intravascular Coagulopathy,Branch Retinal Vein Occlusion, Hypertensive Fundus Changes, OcularIschemic Syndrome, Retinal Arterial Microaneurysms, Coat's Disease,Parafoveal Telangiectasis, Hemi-Retinal Vein Occlusion,Papillophlebitis, Central Retinal Artery Occlusion, Branch RetinalArtery Occlusion, Carotid Artery Disease (CAD), Frosted Branch Angiitis,Sickle Cell Retinopathy and other Hemoglobinopathies, Angioid Streaks,Familial Exudative Vitreoretinopathy; Eales Disease; traumatic, surgicaland environmental disorders, such as, e.g., Sympathetic Ophthalmia,Uveitic Retinal Disease, Retinal Detachment, Trauma, Retinal Laser,Photodynamic therapy, Photocoagulation, Hypoperfusion During Surgery,Radiation Retinopathy, Bone Marrow Transplant Retinopathy; proliferativedisorders, such as, e.g., Proliferative Vitreal Retinopathy andEpiretinal Membranes; infectious disorders, such as, e.g., OcularHistoplasmosis, Ocular Toxocariasis, Presumed Ocular HistoplasmosisSyndrome (POHS), Endophthalmitis, Toxoplasmosis, Retinal DiseasesAssociated with HIV Infection, Choroidal Disease Associate with HIVInfection, Uveitic Disease Associate with HIV Infection, ViralRetinitis, Acute Retinal Necrosis, Progressive Outer Retinal Necrosis,Fungal Retinal Diseases, Ocular Syphilis, Ocular Tuberculosis, DiffuseUnilateral Subacute Neuroretinitis, Myiasis; genetic disorders, such as.e.g., Retinitis Pigmentosa, Systemic Disorders with Accosiated RetinalDystrophies, Congenital Stationary Night Blindness, Cone Dystrophies,Stargardt's Disease And Fundus Flavimaculatus, Best's Disease, PatternDystrophy of the Retinal Pigmented Epithelium, X-Linked Retinoschisis,Sorsby's Fundus Dystrophy, Benign Concentric Maculopathy, Bietti'sCrystalline Dystrophy, pseudoxanthoma elasticum; optic neuropathies,such as, e.g., glaucoma; retinal injuries, such as, e.g., Macular Hole,Giant Retinal Tear; retinal tumors, such as, e.g., Retinal DiseaseAssociated With Tumors, Congenital Hypertrophy Of The RPE, PosteriorUveal Melanoma, Choroidal Hemangioma, Choroidal Osteoma, ChoroidalMetastasis, Combined Hamartoma of the Retina and Retinal PigmentedEpithelium, Retinoblastoma, Vasoproliferative Tumors of the OcularFundus, Retinal Astrocytoma, and Intraocular Lymphoid Tumors.

“Glaucoma” means primary, secondary and/or congenital glaucoma. Primaryglaucoma can include open angle and closed angle glaucoma. Secondaryglaucoma can occur as a complication of a variety of other conditions,such as injury, inflammation, vascular disease and diabetes.

VII. Neurological Conditions and Intracellular Nitric Oxide

The present invention further provides a method for treating aneurological condition associated with abnormal levels of nitric oxideby administering an effective amount of a selective persistent sodiumcurrent antagonist having at least 20-fold selectivity for persistentsodium current relative to transient sodium current. As disclosedherein, selective persistent sodium current antagonists are useful forselectively reducing persistent sodium current, thereby providing aneuroprotective benefit for acute and chronic neuronal insults. Suchantagonists also are useful for reducing deleterious cellular effectsresulting from inappropriately high levels of intracellular nitricoxide, and therefore, can effectively treat conditions characterized byaberrant levels of intracellular nitric oxide. As used herein, the term“condition characterized by aberrant levels of intracellular nitricoxide” means a disorder characterized by amounts of nitric oxide in thecells of an individual, that are increased compared to normal amounts ofnitric oxide. Such excessive amounts of nitric oxide can result, forexample, from excess or unregulated synthesis of nitric oxide.

Nitric oxide is a free radical gas that functions as a signalingmolecule in at least three systems: white blood cells, where nitricoxide mediates tumoricidal and bactericidal effects; blood vessels,where it represents endothelium-derived relaxing factor activity, and inneurons, where it functions much like a neurotransmitter. In addition toits normal role in neurons, nitric oxide can also function as aneurotoxic mediator under pathophysiological conditions. For example,mice having a deletion of the nitric oxide synthase gene were found tobe resistant to focal and transient global ischemia, see, e.g., NarimanPanahian et al., Attenuated Hippocampal Damage After Global Cerebralschemia in Mice Mutant in Neuronal Nitric Oxide Synthase, 72(2)NEUROSCIENCE 343-354 (1996), which is hereby incorporated by referencein its entirety. Therefore, without wishing to be bound by thefollowing, nitric oxide can cause neuronal death by activatingpersistent sodium channels and causing intracellular calcium overload.As non-limiting examples, conditions characterized by aberrant levels ofintracellular nitric oxide include vascular shock, stroke, diabetes,neurodegeneration, asthma, arthritis and chronic inflammation, see,e.g., Nobuyuki Miyasaka & Yukio Hirata, Nitric Oxide and InflammatoryArthritides, 61(21) LIFE SCI. 2073-2081 (1997); Juan P. Bolanos &Ángeles Almeida, Roles of Nitric Oxide in Brain Hypoxia-Ischemia,1411(2-3) BIOCHIM. BIOPHYS. ACTA. 415-436 (1999); Joel E. Barbato &Edith Tzeng Nitric Oxide and Arterial Disease 40(1) J. VASC. SURG.187-193 (2004); Kevin J. Barnham at al., Neurogegenerative diseases andOxidative Stress, 3(3) NAT. REV. DRUG. DIS. 205-214 (2004); Hossein A.Ghofrani et al., Nitric Oxide Pathway and Phosphodiesterase Inhibitorsin Pulmonary Arterial Hypertension, 43(12 Suppl. S) J. AM. COLL.CARDIOL. 68S-72S (2004); Maria A. Moro et al., Role of Nitric Oxideafter Brain ischaemia, 36(3-4) CELL CALCIUM 265-275 (2004); S A.Mulrennan & A. E. Redington, Nitric Oxide Synthase Inhibition:Therapeutic Potential in Asthma, 3(2) TREAT. RESPIR. MED. 79-88 (2004);Fabio L. M. Ricciardolo et al., Nitric Oxide in Health and Disease ofthe Respiratory System, 84(3) PHYSIOL. REV. 731-765 (2004); and SharmaS. Prabhakar, Role of Nitric Oxide in Diabetic Nephropathy, 24(4) SEMIN.NEPHROL. 333-344 (2004), which are hereby incorporated by reference intheir entirety.

Nitric oxide can cause, neurodegeneration and neurotoxicity viavoltage-gated sodium channels, see, e.g., Garthwaite et al, supra,(1999). For example in the optic nerve, the neurodestructive effects ofnitric oxide donors were shown to be ameliorated by compounds that blockvoltage-gated Na⁺ channels such as TTX, see, e.g., Gita Garthwaite etal, Nitric Oxide Toxicity in CNS White Matter: An in Vitro Study UsingRat Optic Nerve, 109(1) NEUROSCIENCE 145-155 (2000a); and GitaGarthwaite et al., Soluble Guanylyl Cyclase Activator YC-1 ProtectsWhite Matter Axons From Nitric Oxide Toxicity and Metabolic Stress,Probably Through Na(+) Channel Inhibition, 61 (1) MOL. PHARMACOL. 97-104(2000b), which are hereby incorporated by reference in their entirety.Thus blocking voltage-gated Na⁺ channels would appear to be a protectivestrategy against the injurious effects of nitric oxide toxicity.However, the normal rapidly inactivating Na⁺ channels do not appear tobe the targets for this strategy. For example, increases in eitherendogenous or exogenous levels of intracellular nitric oxide generateaberrant persistent sodium currents in central neurons and cardiaccells, see, e.g., Anna K. M. Hammarström & Peter W. Gage, Nitric OxideIncreases Persistent Sodium Current in Rat Hippocampal Neurons, 520(2)J. PHYSIOL. 451-461 (1999); and Gerard P. Ahern et al., Induction ofPersistent Sodium Current by Exogenous and Endogenous Nitric Oxide,275(37) J. BIOL. CHEM. 28810-28815 (2000), which are hereby incorporatedby reference in their entirety. This up-regulation of persistent sodiumcurrent by nitric oxide is independent of guanylate cyclase and thusindependent of cGMP formation, see, e.g., Ahern et al., supra, (2000).As such, nitric oxide may contribute to neurodestruction orneurodegeneration via activating or increasing persistent sodiumcurrent. Blocking persistent sodium current upregulated by nitric oxideshould therefore prevent cellular Na⁺ and subsequent Ca²⁺ overloadassociated with neuronal cell death under pathophysiological conditionswhere this current plays a role. Thus blocking the persistent sodiumcurrent in neurons may afford a neuroprotective benefit in the treatmentacute and chronic neuronal insults including neurodegenerative diseaseswhere nitric oxide is thought to play a neurodestructive role.

An additional advantage of targeting the persistent sodiumchannel/current is that it appears to be the final common effector inthe neurodestructive pathway caused by nitric oxide. For example,activation of NMDA receptors under excitotoxic conditions results inexcess nitric oxide production, see, e.g., Strijbos et al, supra, (1996)that then up-regulates persistent sodium currents, see, e.g., Garthwaiteet al, supra, (2000b). Activation of persistent sodium channels wouldthen lead to further membrane depolarization (leading to furtherglutamate release), elevated intracellular Ca²⁺ (via Ca²⁺ influx throughNMDA receptor channels) and reversal of the sodium/calcium exchanger.Elevation of Ca²⁺ through the NMDA receptor and reverse sodium/calciumexchange would further exacerbate the situation since additional Ca²⁺entry would activate more nitric oxide synthase causing a perniciouscycle of neurodestruction.

It is understood that conditions characterized by aberrant levels ofpersistent sodium current or aberrant levels of intracellular nitricoxide can be identified or confirmed using routine methods, includingmethods described herein. It is also understood that one or moretransient sodium currents also can be increased. Similarly, a level ofintracellular nitric oxide in a cell from a subject having a disease orpathological condition can be compared to a level of intracellularnitric oxide in a cell from a normal or non-diseased subject. Anincreased level of intracellular nitric oxide can typically be observedin at least one cell type of a subject having a condition characterizedby aberrant levels intracellular nitric oxide. Human conditionscharacterized by aberrant levels of persistent sodium current oraberrant levels of intracellular nitric oxide in addition to thesedescribed herein above can be identified by those skilled in the art.

VIII. Selective Persistent Sodium Current Blockers

The methods of the invention involve administering a compound thatselectively reduces persistent sodium current relative to transientsodium current. As used herein, the term “selective,” when used hereinin reference to a compound, such as an antagonist, means a compoundthat, at least one particular dose reduces persistent sodium current atleast 20-fold more than transient sodium current is reduced. Therefore,a compound that selectively reduces persistent sodium current has atleast 20-fold selectively for persistent sodium current relative totransient sodium current, and can have, for example, at least 50-foldselectively for persistent sodium current relative to transient sodiumcurrent, at least 100-fold, at least 200-fold, at least 400-fold, atleast 600-fold, or at least 1000-fold selectively for persistent sodiumcurrent relative to transient sodium current.

As used herein, the term “persistent sodium current” means a sodiumchannel mediated current that is non-transient; that can remain activeduring prolonged depolarization or that activates at voltage morenegative than −60 mV and thus can contribute to hyperexcitability of theneural membrane. Prolonged depolarization refers to depolarization thatoccurs over a time period greater than the time period during which atransient current typically inactivates. As a non-limiting example,prolonged depolarization can occur within a time period greater than thetime period during which the transient current of a sodium channel, suchas Na_(v)1.2, typically inactivates. Therefore, prolonged depolarizationrefers to depolarization that persists for at least 0.002 second, suchas at least 0.01 second, at least 0.1 second and at least 1 second.

A compound that selectively reduces persistent sodium current can be,for example, a persistent sodium channel antagonist. As used herein, theterm “persistent sodium channel antagonist,” means a compound thatinhibits or decreases persistent current mediated through a sodiumchannel by binding to the sodium channel. It is understood that apersistent sodium channel antagonist can act by any antagonisticmechanism, such as by directly binding a persistent sodium channel atthe pore entrance, thereby inhibiting movement of ions through thechannel, or by binding a channel at another site to alter channelconformation and inhibit movement of ions through the channel. Exemplaryselective persistent sodium channel antagonists that represent fourstructural classes of organic molecules are disclosed herein as Formulas1, 2, 3 and 4.

It further is understood that a compound that selectively reducespersistent sodium current can act indirectly, for example, by reducingor down-regulating expression of a persistent sodium channel, forexample, by inactivating a positive regulator of transcription oractivating a negative regulator of transcription, without acorresponding reduction transient sodium channel; by increasing theexpression or activity of a molecule that inactivates or reducespersistent sodium channel function, such as a protease, modifying enzymeor other molecule, without a corresponding reduction in transient sodiumcurrent; or by decreasing the expression or activity of a molecule thattransmits a downstream signal from a persistent sodium current without acorresponding reduction in transient sodium current, for example,without substantially altering the downstream signal from a transientsodium channel.

As disclosed herein, structurally unrelated molecules can have at least20-fold selectivity for reducing persistent sodium current relative totransient sodium current and, therefore, can be useful in the methods ofthe invention. For example, such a compound can be a naturally ornon-naturally occurring macromolecule, such as a peptide,peptidomimetic, nucleic acid, carbohydrate or lipid. The compoundfurther can be an antibody, or antigen-binding fragment thereof such asa monoclonal antibody, humanized antibody, chimeric antibody, minibody,bifunctional antibody, single chain antibody (scFv), variable regionfragment (Fv or Fd), Fab or F(ab)₂. The compound also can be a partiallyor completely synthetic derivative, analog or mimetic of a naturallyoccurring macromolecule, or a small organic or inorganic molecule.

A selective persistent sodium current antagonist that is a nucleic acidcan be, for example, an anti-sense nucleotide sequence, an RNA molecule,or an aptamer sequence. An anti-sense nucleotide sequence can bind to anucleotide sequence within a cell and modulate the level of expressionof a persistent, sodium channel gene, or modulate expression of anothergene that controls the expression or activity of a persistent sodiumchannel. Similarly, an RNA molecule, such as a catalytic ribozyme, canbind to and alter the expression of a persistent sodium channel gene, orother gene that controls the expression or activity of a persistentsodium channel. An aptamer is a nucleic acid sequence that has a threedimensional structure capable of binding to a molecular target, see,e.g., Sumedha D. Jayasena, Aptamers: An Emerging Class of Molecules ThatRival Antibodies in Diagnostics, 45(9) CLIN. CHEM. 1628-1650 (1999),which is hereby incorporated by reference in its entirety. As such, anaptamer can serve as a persistent sodium current selective compound.

A selective persistent sodium current antagonist that is a nucleic acidalso can be a double-stranded RNA molecule for use in RNA interferencemethods. RNA interference (RNAi) is a process of sequence-specific genesilencing by post-transcriptional RNA degradation, which is initiated bydouble-stranded RNA (dsRNA) homologous in sequence to the silenced gene.A suitable double-stranded RNA (dsRNA) for RNAi contains sense andantisense strands of about 21 contiguous nucleotides corresponding tothe gene to be targeted that form 19 RNA base pairs, leaving overhangsof two nucleotides at each 3′ end (Sayda M. Elbashir et al., Duplexes of21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells,411(6836) NATURE 494-498 (2001); B. L. Bass, RNA Interference. The ShortAnswer, 411(6836) NATURE 428-429 (2001); Phillip D. Zamore, RNAInterference: Listening to the Sound of Silence, 8(9) NAT. STRUCT. BIOL.746-750 (2001), which are hereby incorporated by reference in theirentirety. dsRNAs of about 25-30 nucleotides have also been usedsuccessfully for RNAi (Anton Karabinos et al., Essential Roles for FourCytoplasmic Intermediate Filament Proteins in Caenorhabditis elegansDevelopment, 98(14) PROC. NATL. ACAD. SCI. USA 7863-7868 (2001), whichis hereby incorporated by reference in its entirety. dsRNA can besynthesized in vitro and introduced into a cell by methods known in theart.

A persistent sodium channel selective compound that is an antibody canbe, for example, an antibody that binds to a persistent sodium channeland inhibits movement of ions through the channel, or alters theactivity of a molecule that regulates persistent sodium currentexpression or activity, such that sodium current is decreased. It isunderstood that such a compound binds selectively such that acorresponding reduction in transient sodium current is not affected.

A persistent sodium channel selective compound that is a small moleculecan have a variety of structures. In several embodiments, a compoundthat selectively reduces persistent sodium current that has at least20-fold selectivity for reducing persistent sodium current tonon-persistent sodium current is an organic molecule represented by aformula shown herein below, or a pharmaceutically acceptable salt,ester, amide, steroisomer or racemic mixture thereof. As disclosedherein in FIG. 1, several identified compounds are selective forpersistent sodium current relative to transient sodium current, withselectivities of 32-fold, 38-fold, 110-fold and 453-fold. It isunderstood that these and other compounds with at least 20-foldselectivity for persistent sodium current relative to transient sodiumcurrent, for example, identified by the methods disclosed herein inExamples 1, 2, 3 and 4 can be useful for treating a neurologicaldisorder according to a method of the invention.

In one embodiment, a compound useful in a method of the invention, or apharmaceutically acceptable salt, ester, amide, stereoisomer or racemicmixture thereof, has a structure from Formula 1:

wherein,

Ar¹ is an aryl group;

Ar² is an aryl group;

Y is absent or is selected from:

R¹ is selected from hydrogen, C₁-C₈ alkyl, aryl, or arylalkyl;

R² and R³ are independently selected from hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy, fluoro, C₁-C₈ carbocyclic ring, or C₁-C₈heterocyclic ring;

R⁴ is selected from hydrogen, C₁-C₈ alkyl, aryl, or arylalkyl;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, orhydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, or arylalkyl, and

n is an integer of from 1 to 6.

In one aspect of this embodiment, Ar¹ is thienyl, or substitutedthienyl. For example, the thienyl can be substituted with one or more ofhalogen, C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5; and

In another aspect of this embodiment, Ar² is phenyl or substitutedphenyl. For example, the phenyl can be substituted with halogen, C₁-C₈alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN and (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5.

In another embodiment, a compound useful in a method of the invention,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orracemic mixture thereof, has a structure from Formula 4:

wherein,

Ar³ is an aryl group;

Ar⁴ is an aryl group;

X¹ and Y¹ are independently selected from:

R⁵ and R⁶ are independently selected from: hydrogen, fluoro, C₁ to C₈alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R⁸ and R⁹ are selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl,COR¹², COCF₃;

R¹⁰ and R¹¹ are selected from hydrogen, halogen, hydroxyl, C₁-C₈ alkyl,aryl, arylalkyl; and

R¹² is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl.

In one aspect of this embodiment, Ar³ can be phenyl or substitutedphenyl. For example, the phenyl can be substituted with one or more ofhalogen, C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)CN(R⁷)₂, whereinc is 0 or an integer from 1 to 5.

In another aspect of this embodiment, Ar⁴ is substituted with one ormore of halogen, C₁-C₈ alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN or(CR⁵R⁶)_(c)N(R⁷)₂, wherein c is 0 or an integer from 1 to 5.

In yet another embodiment, a compound useful in a method of theinvention, or a pharmaceutically acceptable salt, ester, amide,stereoisomer or racemic mixture thereof, has a structure from Formula 2:

wherein,

Ar⁵ is an aryl group;

Ar⁶ is an aryl group;

X² is O, S, or NR¹⁴;

Y² is N or CR¹⁵;

Z² is N or CR¹⁶;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹³ is selected from halogen, C₁-C₈ alkyl, arylalkyl, and(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁴ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁵ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁶ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂, and

wherein c is 0 or an integer from 1 to 5.

In one aspect of this embodiment, Ar⁵ is phenyl or substituted phenyl.For example, the phenyl can be substituted with one or more of halogen,C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, or (CR⁵R⁶)_(c)N(R⁷)₂, wherein cis 0 or an integer from 1 to 5.

In another aspect of this embodiment, Ar⁶ is substituted with halogen,C₁-C₈ alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN or (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5.

In yet another aspect of this embodiment, Ar⁶ is selected from:

In yet another embodiment, a compound useful in a method of theinvention, or a pharmaceutically acceptable salt, ester, amide,stereoisomer or racemic mixture thereof, has a structure from Formula 3:

wherein,

Ar⁷ is an aryl group;

R_(a) is selected from halogen, C₁-C₈ alkyl, NR²²R²³, OR²²;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹⁷ and R¹⁸ are independently selected hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, and hydroxy;

R¹⁹ and R²⁰ are independently selected from hydrogen, halogen, C₁-C₈alkyl, hydroxy, amino, CF₃;

R²¹, R²², and R²³ are independently selected from hydrogen, aryl orC₁-C₈ alkyl;

a is 0 or an integer from, 1 to 5, and

m is 0 or and integer from 1 to 3.

In one aspect of this embodiment, Ar⁷ is phenyl or substituted phenyl.For example the phenyl can be substituted with one or more of halogen,C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)_(c)N(R⁷)₂, wherein c is0 or an integer from 1 to 5.

In another aspect of this embodiment, R is amino or

In yet another aspect of this embodiment, R¹⁷ is isopropyl; in oneembodiment, R¹⁸ is methyl.

Exemplary compounds that are persistent sodium channel antagonistsuseful in a method of the invention are shown as Formulas 1, 2, 3 and 4.In addition, the compounds shown in FIG. 1 have selectivities forpersistent sodium current of 32-fold, 38-fold, 110-fold, and 453-fold,relative to transient sodium current.

As used herein, the term “alkyl” means a straight-chain, branched orcyclic saturated aliphatic hydrocarbon. For example, an alkyl group canhave 1 to 12 carbons, such as from 1 to 7 carbons, or from 1 to 4carbons. Exemplary alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.An alkyl group may be optionally substituted with one or moresubstituents are selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino, and SH.

As used herein, the term “alkenyl” means a straight-chain, branched orcyclic unsaturated hydrocarbon group containing at least onecarbon-carbon double bond. For example, an alkenyl group can have 1 to12 carbons, such as from 1 to 7 carbons, or from 1 to 4 carbons. Analkenyl group can optionally be substituted with one or moresubstituents. Exemplary substituents include hydroxyl, cyano, alkoxy,═O, ═S, NO₂, halogen, dimethyl amino, and SH.

As used herein, the term “alkynyl” means a straight-chain, branched orcyclic unsaturated hydrocarbon containing at least one carbon-carbontriple bond. For example, an alkynyl group can have 1 to 12 carbons,such as from 1 to 7 carbons, or from 1 to 4 carbons. An alkynyl groupcan optionally be substituted With one or more substituents. Exemplarysubstituents include hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen,dimethyl amino, and SH.

As used herein, the term “alkoxyl” means an “O-alkyl” group.

As used herein, the term “aryl” means an aromatic group which has atleast one ring having a conjugated pi electron system and includescarbocyclic aryl, heterocyclic aryl and biaryl groups. An aryl group canoptionally be substituted with one or more subtituents. Exemplarysubstituents include halogen, trihalomethyl, hydroxyl, SH, OH, NO₂,amine, thioether, cyano, alkoxy, alkyl, and amino.

As used herein, the term “alkaryl” means an alkyl that is covalentlyjoined to an aryl group. The alkyl can be, for example, a lower alkyl.

As used herein, the term “carbocyclic aryl” means an aryl group whereinthe ring atoms are carbon.

As used herein, the term “heterocyclic aryl” means an aryl group havingfrom 1 to 3 heteroatoms as ring atoms, the remainder of the ring atomsbeing carbon. Heteroatoms include oxygen, sulfur, and nitrogen. Thus,heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl,N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.

As used herein, the term “hydrocarbyl” means a hydrocarbon radicalhaving only carbon and hydrogen atoms. For example, an hydrocarbylradical can have from 1 to 20 carbon atoms, such as from 1 to 12 carbonatoms or from 1 to 7 carbon atoms.

As used herein, the term “substituted hydrocarbyl” means a hydrocarbylradical wherein one or more, but not all, of the hydrogen and/or thecarbon atoms are replaced by a halogen, nitrogen, oxygen, sulfur orphosphorus atom or a radical including a halogen, nitrogen, oxygen,sulfur or phosphorus atom, e.g. fluoro, chloro, cyano, nitro, hydroxyl,phosphate, thiol, etc.

As used herein, the term “amide” means —C(O)—NH—R′, wherein R′ is alkyl,aryl, alkylaryl or hydrogen. As used herein, the term “thioamide” means—C(S)—NH—R′, wherein R′ is alkyl, aryl, alkylaryl or hydrogen. As usedherein, the term “amine” means a —N(R″)R′″ group, wherein R″ and R′″ areindependently selected from the group consisting of alkyl, aryl, andalkylaryl. As used herein, the term “thioether” means —S—R″, wherein R″is alkyl, aryl, or alkylaryl. As used herein, the term “sulfonyl” refersto —S(O)₂—R″″, where R″″ is aryl, C(CN)═C-aryl, CH₂CN, alkyaryl,sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl.

IX. Screening Assays

The ability of a compound to selectively reduce persistent sodiumcurrent relative to transient sodium current can be determined using avariety of assays. Such assays can be performed, for example, in a cellor tissue that expresses an endogenous or recombinantly expressedpersistent sodium current, and generally involve determining persistentand transient sodium current prior to and following application of atest compound.

Methods for measuring sodium current are well known to those skilled inthe art, and are described, see, e.g., Joseph S. Adorante, Inhibition ofNoninactivating Na Channels of Mammalian Optic Nerve as a Means ofPreventing Optic Nerve Degeneration Associated with Glaucoma, U.S. Pat.No. 5,922,746 (Jul. 13, 1999); Bert Sakmann & Erwin Neher, SINGLECHANNEL RECORDING (Plenum Press, 2^(nd) ed. 1995); and Tsung-Ming Shihet al., High-level Expression and Detection of Ion Channels in XenopusOocytes, 529-556 (METHODS IN ENZYMOLOGY: ION CHANNELS PART B, Vol. 293,P. Michael Conn ed., Academic Press 1998), which are hereby incorporatedby reference in their entirety. These protocols are routine procedureswell within the scope of one skilled in the art and from the teachingherein (see, e.g., Examples 1, 2, 3 and 4). Since the rate at whichsodium currents open and close is rapid and the speed at which ionsflow, through the channel is high, channel function can be studied usingan electrophysiological approach, which is capable of measuring the ionflux at the rate of one million ions per second with a millisecond timeresolution. In addition, as shown in Examples 1, 2 and 3, a method foridentifying a selective persistent sodium channel antagonist or otherpersistent sodium current antagonist can involve using a fluorescent dyethat is sensitive to change in cell membrane potential in order toenable optical measurement of cell membrane potential. As disclosedherein below, a compound to be tested is added to a well containingcells that express a sodium channel capable of mediating a persistentsodium current, and express a potassium channel or a sodium/potassiumATPase or both.

Methods for measuring membrane potential with voltage-sensitive dyes arewell known to those skilled in the art, and are described, see, e.g.,lain D. Johnson, Fluorescent Probes for Living Cells 30(3) HISTOCHEM. J.123-140 (1998); and IMAGING NEURONS: A LABORATORY MANUAL (Rafael Yuste,et al., eds., Cold Spring Harbor Laboratory Press, 2000). In particular,the example listed below takes advantage of the high temporal andspatial resolution that derives from utilization of fluorescenceresonance energy transfer (FRET) in the measurement of membranepotential by voltage-sensitive dyes as described, see, e.g., Jesus E.Gonzalez & Roger Y. Tsien, Improved Indicators of Cell MembranePotential That Use Fluorescence Resonance Energy Transfer 4(4) CHEM.BIOL. 269-277 (1997); Roger Y. Tsien & Jesus E. Gonzalez, VoltageSensing by Fluorescence Resonance Energy Transfer, U.S. Pat. No.5,661,035 (Aug. 26, 1997); Roger Y. Tsien & Jesus E. Gonzalez, Detectionof Transmembrane Potentials by Optical Methods, U.S. Pat. No. 6,342,379(Jan. 29, 2002); Jesus E. Gonzalez & Michael P. Maher, CellularFluorescent Indicators and Voltage/Ion Probe Reader (VIPR) Tools for IonChannel and Receptor Drug Discovery, 8(5-6) RECEPTORS CHANNELS 283-295,(2002); and Michael P. Maher & Jesus E. Gonzalez, High Throughput Methodand System for Screening Candidate Compounds for Activity Against TargetIon Channels, U.S. Pat. No. 6,686,193 (Feb. 3, 2004), which are herebyincorporated by reference in their entirety.

In addition, the selectivity of a compound for persistent sodium currentversus transient sodium current can be confirmed, as shown in theteaching herein (see, e.g., Examples 2 and 3).

A variety of cell types, including naturally occurring cells andgenetically engineered cells can be used in an in vitro assay to detectpersistent sodium current. Naturally occurring cells havingnon-inactivating sodium current include, for example, several types ofneurons, such as squid axon, cerebellar Purkinje cells, neocorticalpyramidal cells, thalamic neurons, CA1 hipppocampal pyramidal cells,striatal neurons and mammalian CNS axons. Other naturally occurringcells having persistent sodium current can be identified by thoseskilled in the art using methods disclosed herein below and other wellknown methods. Cells for use in testing a compound for its ability toalter persistent sodium current can be obtained from a mammal, such as amouse, rat, pig, goat, monkey or human, or a non-mammal containing acell expressing a sodium channel capable of mediating persistent sodiumcurrent.

Genetically engineered cells having persistent sodium current cancontain, for example, a cDNA encoding a sodium channel capable ofmediating a persistent current such as Na_(v)1.3; or can be a cellengineered to have increased expression of a sodium channel capable ofmediating a persistent current, decreased expression of a sodium channelmediating a transient current, or both. Recombinant expression isadvantageous in providing a higher level of expression of a sodiumchannel capable of mediating a persistent sodium current than is foundendogenously and also allows expression in cells or extracts in whichthe channel is not normally found. One or more recombinant nucleic acidexpression constructs generally contain a constitutive or induciblepromoter of RNA transcription appropriate for the host cell ortranscription-translation system, operatively linked to a nucleotidesequence that encodes one or more polypeptides of the channel ofinterest. The expression construct can be DNA or RNA, and optionally canbe contained in a vector, such as a plasmid or viral vector. Based onwell-known and publicly available knowledge of nucleic acid sequencesencoding subunits of many sodium channels, including several sodiumchannels capable of mediating a persistent sodium current, one skilledin the art can express desired levels of a biologically activepersistent or transient sodium channels using routine laboratory methodsas described, see, e.g., Molecular Cloning A Laboratory Manual (JosephSambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press,3^(rd) ed. 2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FrederickM. Ausubel et al., eds., John Wiley & Sons, 2004), which are herebyincorporated by reference in their entirety. cDNAs for several familiesof sodium channels have been cloned and sequenced, and are described,see, e.g, Alan L. Goldin, Diversity of Mammalian Voltage-gated SodiumChannels, 868 ANN. N.Y. ACAD. SCI. 38-50 (1999), William A. Catterall,From Ionic Currents to Molecular Mechanisms: The Structure and Functionof Voltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); John N.Wood & Mark D. Baker, Voltage-gated Sodium Channels, 1(1) CURR. OPIN.PHARMACOL. 17-21 (2001); and Frank H. Yu & William A. Catterall,Overview of the Voltage-Gated Sodium Channel Family, 4(3) GENOME BIOL.207 (2003), which are hereby incorporated by reference in theirentirety. In addition, both nucleotide and protein sequences allcurrently described sodium channels are publicly available from theGenBank database (National Institutes of Health, National Library ofMedicine, http://www.ncbi.nlm.nih.gov/), which is hereby incorporated byreference in its entirety.

Exemplary host cells that can be used to express recombinant sodiumchannels include isolated mammalian primary cells; established mammaliancell lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12;amphibian cells, such as Xenopus embryos and oocytes; and othervertebrate cells. Exemplary host cells also include insect cells such asDrosophila, yeast cells such as S. cerevisiae, S. pombe, or Pichiapastoris and prokaryotic cells (such as E. coli,) engineered torecombinantly express sodium channels.

X. Reaction Schemes

A compound used in a method of the invention can be synthesized bygeneral synthetic methodology, such as by the specific syntheticreaction schemes and methodologies described below and in Examples 5, 6,7 and 8. Modifications of these synthetic methodologies will becomereadily apparent to the practicing synthetic organic chemist in view ofthe following disclosure and general knowledge available in the art.

The reaction schemes disclosed below are directed to the synthesis ofexemplary compounds used in a method of the invention. The syntheticprocesses described herein are adaptable within the skill of thepracticing organic chemist and can be used with such adaptation for thesynthesis of compounds useful in a method of the invention that are notspecifically described. Reaction schemes 1, 2, 3 and 4 disclosesynthetic routes to compounds having Formulas 1, 2, 3 and 4,respectively. Examples 5, 6, 7 and 8 describe methodology useful forsynthesizing exemplary compounds representative of Formulas 1, 2, 3 and4, respectively.

The specific reaction conditions described in Examples 5, 6, 7 and 8 aredirected to the synthesis of exemplary compounds useful in a method ofthe invention. Whereas each of the specific and exemplary syntheticmethods shown in Examples 5, 6, 7 and 8 describe specific compoundswithin the scope of general Formulas 1 through 4, the syntheticprocesses and methods used therein are adaptable within the skill of thepracticing organic chemist and can be used with such adaptation for thesynthesis of compounds useful in a method of the invention that are notspecifically described herein as examples.

XI. Animal Models

The efficacy of a compound that selectively reduces persistent sodiumcurrent, such as a selective persistent sodium current antagonist, intreating a condition characterized by aberrant levels of sodium currentor aberrant levels of intracellular nitric oxide in a mammal can beconfirmed using a variety of well-known methods. Well-known animalmodels can be useful for determining the ability of a compound, such asa selective persistent sodium current antagonist, to reduce neuronaldeath or treat a condition characterized by aberrant levels of sodiumcurrent or condition characterized by aberrant levels of intracellularnitric oxide. Ischemia can be induced in several animal species usingany of several surgical procedures, which can employ, for example, anyof intralumenal occlusions, extralumenal occlusions, vascular clips,miniature hydraulic occluders or Ameroid occluders. Specific animalmodels of ischemia are well known to those skilled in the art, andexemplary ischemia models including rodent, monkey, baboon, dog, gerbiland rabbit models are described, see, e.g., Douglas E. McBean & Paul A.T. Kelly, Rodent Models of Global Cerebral Ischemia: A Comparison ofTwo-Vessel Occlusion and Four-Vessel Occlusion 30(4) GEN. PHARMACOL.431-434 (1998); E. M. Nemoto, Monkey Model of Complete Global Ischemia,24(2) STROKE 328-329 (1993); R. F. Spetzler et al., Chronic ReversibleCerebral Ischemia: Evaluation of a New Baboon Model, 7(3) NEUROSURGERY257-261 (1980); A. Mitro et al., Method of the Development ofIrreversible, Complete Cerebral Ischemia in Dog, 21(2) NEUROPATOL. POL.315-321 (1983); T. Yoshimine & T Yanagihara, Regional Cerebral Ischemiaby Occlusion of the Posterior Communicating Artery and the MiddleCerebral Artery in Gerbils, 58(3) J. NEUROSURG. 362-367 (1983); R.Pluta, Experimental Treatment with Prostacyclin of Global CerebralIschemia in Rabbit-New Data, 28(3-4) NEUROPATOL. POL. 205-215 (1990); J.Guo & Y. D. Chao, Modification of a Model for Cerebral Ischemia in theCat: A New Method to Occlude the Middle Cerebral Artery, 25(1)NEUROSURGERY 49-53 (1989).

Animal models of neurodegenerative disorders are well known in the art,and include, for example, include Alzheimer's disease models, such astransgenic mice over-expressing mutant forms of amyloid precursorprotein and presenilin-1, and models in which animals treated withamyloid β-peptide or excitotoxins. An exemplary model of Parkinson'sdisease involves administration of the toxin MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to animals, such asmonkeys and mice, which results in selective loss of substantia nigradopaminergic neurons and associated motor dysfunction. Neurodegenerativedisease models can employ a variety of animals including, but notlimited to, mice, gerbils, rats, rabbits, pigs, cats, dogs, sheep andprimates, see, e.g., Senile Dementia of Alzheimer Type: Early Diagnosis,Neuropathology and Animal Models (J Traber & W. H. Gispen, eds.,Springer Verlag, 1985); CENTRAL NERVOUS SYSTEM DISEASES: INNOVATIVEANIMAL MODELS FROM LAB TO CLINIC (Dwaine F. Emerich et al., eds., HumanaPress, 1999).

End-points useful for assessing the effect of a compound thatselectively reduces persistent sodium current, such as a selectivepersistent sodium current antagonist, on the extent of neuronal death ordysfunction in comparison to a control animal that has not received thecompound depend, in part, on the condition to be treated and are wellknown to those skilled in the art. Such end points included, forexample, reduction in lesion size, improved physiological function, andimproved behavior.

The activity of a compound that selectively reduces persistent sodiumcurrent, such as a selective persistent sodium current antagonist, alsocan be confirmed in a cell-based model of neuronal damage. Such acell-based model can provide another read-out for the activity of anantagonist prior to its use in an animal model, and can also be used toidentify antagonists or other compounds useful for reducing death ofcultured neurons. Exemplary cell-based assays include cell models ofischemia-induced neuronal damage in which neurons demonstrate one ormore indicia of apoptosis in response to a substance or condition, suchas hypoxia, glucose deprivation, oxidative or excitotoxic insult.Exemplary cell-based models of ischemia-induced neuronal damage areknown to those of skill in the art and are described, for example, inLalitha Tenneti et al., Role of Caspases in N-Methyl-D-Aspartate-InducedApoptosis in Cerebrocortical Neurons, 71(3) J. NEUROCHEM. 946-959(1998); and R. James White & Ian J. Reynolds, MitochondrialDepolarization in Glutamate-Stimulated Neurons: An Early Signal Specificto Excitotoxin Exposure, 16(18) J. NEUROSCI. 5688-5697 (1996).

The ability of a compound that selectively reduces persistent sodiumcurrent, such as a selective persistent sodium current antagonist, toreduce neuronal death or dysfunction can be assessed by analyzing anobservable sign or symptom of nerve cell destruction in the presence andabsence of treatment with the compound. Initiation of apoptotic death ofneurons can have observable effects on cell function and morphology, aswell as observable effects on tissues, organs and animals that containdysfunctional or apoptotic neurons. Therefore, an indicator of neuronaldamage can include observable parameters of molecular changes, such asincreased expression of apoptosis-induced genes; cell function changes,such as reduced mitochondrial functions; cell morphological changes,such as cell shrinkage and blebbing; organ and tissue functional andmorphological changes, such as the presence of an infarct or otherlesion, the severity of which can be assessed by parameters includinglesion volume and lesion size; physiological changes in animal models,including functional changes, such as loss of motor function, increasedmortality and decreased survival, and behavioral changes, such as onsetof dementia or loss of memory.

A reduction in an indicator of neuronal damage can be assessed in acell, tissue, organ or animal by comparing an indicator of neuronaldamage in at least two states of a cell, tissue, organ or animal. Thus,a reduction in an indicator of neuronal damage can be expressed relativeto a control condition. A control condition can be, for example, a cell,tissue, organ or animal prior to treatment, in the absence of treatment,in the presence of a different treatment, in a normal animal or anothercondition determined to be appropriate by one skilled in the art.

XII. Pharmaceutical Compositions

As disclosed herein a selective persistent sodium current antagonist isadministered to a mammal to treat a condition characterized by aberrantlevels of sodium current or aberrant levels of intracellular nitricoxide. As used herein, the term “treating,” when used in reference toadministering to a mammal an effective amount of a selective persistentsodium current antagonist, means reducing a symptom of a conditioncharacterized by aberrant levels of sodium current or aberrant levels ofintracellular nitric oxide, or delaying or preventing onset of a symptomof a condition characterized by aberrant levels of sodium current oraberrant levels of intracellular nitric oxide in the mammal. Forexample, the term “treating” can mean reducing a symptom of a conditioncharacterized by aberrant levels of sodium current or aberrant levels ofintracellular nitric oxide by at least 30%, 40%, 60%, 70%, 80%, 90% or100%. The effectiveness of a selective persistent sodium currentantagonist in treating a condition characterized by aberrant levels ofsodium current or aberrant levels of intracellular nitric oxide can bedetermined by observing one or more clinical symptoms or physiologicalindicators associated with the condition. An improvement in a conditioncharacterized by aberrant levels of sodium current or aberrant levels ofintracellular nitric oxide also can be indicated by a reduced need for aconcurrent therapy. Those of skill in the art will know the appropriatesymptoms or indicators associated with specific conditions and will knowhow to determine if an individual is a candidate for treatment with aselective persistent sodium current antagonist. In particular, it isunderstood that those skilled in the art will be able to determine if acondition if characterized by aberrant persistent sodium current, forexample, by comparison of levels of persistent sodium channel expressionor activity in cells from the individual with a normal control cells.

The appropriate effective amount to be administered for a particularapplication of the methods can be determined by those skilled in theart, using the guidance provided herein. For example, an effectiveamount can be extrapolated from in vitro and in vivo assays as describedherein above. One skilled in the art will recognize that the conditionof the patient can be monitored throughout the course of therapy andthat the effective amount of a selective persistent sodium currentantagonist that is administered can be adjusted accordingly.

The invention also can be practiced by administering an effective amountof persistent sodium current antagonist together with one or more otheragents including, but not limited to, one or more analgesic agents. Insuch “combination” therapy, it is understood that the antagonist can bedelivered independently or simultaneously, in the same or differentpharmaceutical compositions, and by the same or different routes ofadministration as the one or more other agents.

Exemplary compounds that have at least 20-fold selectivity for reducingpersistent sodium current relative to non-persistent sodium currentinclude those shown in Formulas 1, 2, 3 and 4. Also encompassed by theinvention are pharmaceutically acceptable salts, esters and amidesderived from Formulas 1, 2, 3 or 4. Suitable pharmaceutically acceptablesalts of the antagonists useful in the invention include, withoutlimitation, acid addition salts, which can be formed, for example, bymixing a solution of the antagonist with a solution of an appropriateacid such as hydrochloric acid, sulfuric acid, fumaric acid, maleicacid, succinic acid, acetic acid, benzoic acid, citric acid, tartaricacid, carbonic acid or phosphoric acid. Where an antagonist carries anacidic moiety, suitable pharmaceutically acceptable salts thereof caninclude alkali salts such as sodium or potassium salts; alkaline earthsalts such as calcium or magnesium salts; and salts formed with suitableorganic ligands, for example, quaternary ammonium salts. Representativepharmaceutically acceptable salts include, yet are not limited to,acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,borate, bromide, calcium edetate, camsylate, carbonate, chloride,clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate,tosylate, triethiodide and valerate.

Thus, it is understood that the functional groups of antagonists usefulin the invention can be modified to enhance the pharmacological utilityof the compound. Such modifications are well within the knowledge of theskilled chemist and include, without limitation, esters, amides, ethers,N-oxides, and pro-drugs of the indicated antagonist. Examples ofmodifications that can enhance the activity of an antagonist include,for example, esterification such as the formation of C1 to C6 alkylesters, such as C1 to C4 alkyl esters, wherein the alkyl group is astraight or branched chain. Other acceptable esters include, forexample, C5 to C7 cycloalkyl esters and arylalkyl esters such as benzylesters. Such esters can be prepared from the compounds described hereinusing conventional methods well known in the art of organic chemistry.

Other pharmaceutically acceptable modifications include the formation ofamides. Useful amide modifications include, for example, those derivedfrom ammonia; primary C1 to C6 dialkyl amines, where the alkyl groupsare straight or branched chain; and arylamines having varioussubstitutions. In the case of secondary amines, the amine also can be inthe form of a 5- or 6-member ring. Methods for preparing these and otheramides are well known in the art.

It is understood that, where an antagonist useful in the invention hasat least one chiral center, the antagonist can exist as chemicallydistinct enantiomers. In addition, where an antagonist has two or morechiral centers, the compound exists as diastereomers. All such isomersand mixtures thereof are encompassed within the scope of the indicatedantagonist. Similarly, where an antagonist possesses a structuralarrangement that permits the structure to exist as tautomers, suchtautomers are encompassed within the scope of the indicated antagonist.Furthermore, in crystalline form, an antagonist can exist as polymorphs;in the presence of a solvent, an antagonist can form a solvate, forexample, with water or a common organic solvent. Such polymorphs,hydrates and other solvates also are encompassed within the scope of theindicated antagonist as defined herein.

A selective persistent sodium current antagonist or other compounduseful in the invention generally is administered in a pharmaceuticalacceptable composition. As used herein, the term “pharmaceuticallyacceptable” refer to any molecular entity or composition that does notproduce an adverse, allergic or other untoward or unwanted reaction whenadministered to a human or other mammal. As used herein, the term“pharmaceutically acceptable composition” refers to a therapeuticallyeffective concentration of an active ingredient. A pharmaceuticalcomposition may be administered to a patient alone, or in combinationwith other supplementary active ingredients, agents, drugs or hormones.The pharmaceutical compositions may be manufactured using any of avariety of processes, including, without limitation, conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

It is also envisioned that a pharmaceutical composition disclosed in thepresent specification can optionally include a pharmaceuticallyacceptable carriers that facilitate processing of an active ingredientinto pharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, auxiliary or excipient.” Such a carriergenerally is mixed with an active compound, or permitted to dilute orenclose the active compound and can be a solid, semi-solid, or liquidagent. It is understood that the active ingredients can be soluble orcan be delivered as a suspension in the desired carrier or diluent. Anyof a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., distilled,deionized water, saline; solvents; dispersion media; coatings;antibacterial and antifungal agents; isotonic and absorption delayingagents; or any other inactive ingredient. Selection of apharmacologically acceptable carrier can depend on the mode ofadministration. Except insofar as any pharmacologically acceptablecarrier is incompatible with the active ingredient, its use inpharmaceutically acceptable compositions is contemplated. Non-limitingexamples of specific uses of such pharmaceutical carriers can be foundin PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C.Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20^(th) ed. 2000); GOODMAN& GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and HANDBOOK OFPHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003) which are hereby incorporated by reference in theirentirety. These protocols are routine procedures and any modificationsare well within the scope of one skilled in the art and from theteaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE®.Tonicity adjustors useful in a pharmaceutical composition include,without limitation, salts such as, e.g., sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjustor. The pharmaceutical composition may be provided as asalt and can be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. It is understood that these andother substances known in the art of pharmacology can be included in apharmaceutical composition useful in the invention.

An antagonist useful in a method of the invention is administered to amammal in an effective amount. Such an effective amount generally is theminimum dose necessary to achieve the desired therapeutic effect, whichcan be, for example, that amount roughly necessary to reduce thesymptoms associated with a neurological disorder, such as, e.g.,epilepsy, cerebral hypoxia, cardiac ischemia, multiple sclerosis andamyotrophic lateral sclerosis. For example, the term “effective amount”when used with respect to treating a neurological disorder can be a dosesufficient to the symptoms, for example, by at least 30%, 40%, 50%, 60%,70%, 80%, 90% or 100%. Such a dose generally is in the range of 0.1-1000mg/day and can be, for example, in the range of 0.1-500 mg/day, 0.5-500mg/day, 0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or0.5-5 mg/day, with the actual amount to be administered determined by aphysician taking into account the relevant circumstances including theseverity of the neurological disorder, the age and weight of thepatient, the patient's general physical condition, the cause of theneurological disorder and the route of administration. Where repeatedadministration is used, the frequency of administration depends, inpart, on the half-life of the antagonist. Suppositories and extendedrelease formulations can be useful in the invention and include, forexample, dermal patches, formulations for deposit on or under the skinand formulations for intramuscular injection. It is understood thatslow-release formulations also can be useful in the methods of theinvention. The subject receiving the selective persistent sodium channelantagonist can be any mammal or other vertebrate capable of experiencinga neurological disorder, for example, a human, primate, horse, cow, dog,cat or bird.

Various routes of administration can be useful for treating aneurological disorder, such as, e.g., epilepsy, cerebral hypoxia,cardiac ischemia, multiple sclerosis and amyotrophic lateral sclerosis,according to a method of the invention. A pharmaceutical compositionuseful in the methods of the invention can be administered to a mammalby any of a variety of means depending, for example, on the type andlocation of a neurological disorder to be treated, the antagonist orother compound to be included in the composition, and the history, riskfactors and symptoms of the subject. Routes of administration suitablefor the methods of the invention include both systemic and localadministration. As non-limiting examples, a pharmaceutical compositionuseful for treating a neurological disorder can be administered orallyor by subcutaneous pump; by dermal patch; by intravenous, subcutaneousor intramuscular injection; by topical drops, creams, gels or ointments;as an implanted or injected extended release formulation; as abioerodible or non-bioerodible delivery system; by subcutaneous minipumpor other implanted device; by intrathecal pump or injection; or byepidural injection. An exemplary list of biodegradable polymers andmethods of use are described in, e.g., Heller, Biodegradable Polymers inControlled Drug Delivery (CRC CRITICAL REVIEWS IN THERAPEUTIC DRUGCARRIER SYSTEMS, Vol. 1. CRC Press, 1987); Vernon G. Wong, Method forReducing or Preventing Transplant Rejection in the Eye and IntraocularImplants for Use Therefor, U.S. Pat. No. 6,699,493 (Mar. 2, 2004);Vernon G. Wong & Mae W. L. Hu, Methods for TreatingInflammation-mediated Conditions of the Eye, U.S. Pat. No. 6,726,918(Apr. 27, 2004); David A. Weber et al., Methods and Apparatus forDelivery of Ocular Implants, U.S. Patent Publication No. US2004/0054374(Mar. 18, 2004); Thierry Nivaggioli et al., Biodegradable OcularImplant, U.S. Patent Publication No. US2004/0137059 (Jul. 15, 2004),which are hereby incorporated by reference in their entirety. It isunderstood that the frequency and duration of dosing will be dependent,in part, on the relief desired and the half-life of the selectivepersistent sodium current antagonist.

In particular embodiments, a method of the invention is practiced byperipheral administration of a selective persistent sodium currentantagonist. As used herein, the term “peripheral administration” or“administered peripherally” means introducing an agent into a subjectoutside of the central nervous system. Peripheral administrationencompasses any route of administration other than direct administrationto the spine or brain. As such, it is clear that intrathecal andepidural administration as well as cranial injection or implantation arenot within the scope of the term “peripheral administration” or“administered peripherally.” It further is clear that some selectivepersistent sodium current antagonists can cross the blood-brain barrierand, thus, become distributed throughout the central and peripheralnervous systems following peripheral administration.

Peripheral administration can be local or systemic. Local administrationresults in significantly more of a pharmaceutical composition beingdelivered to and about the site of local administration than to regionsdistal to the site of administration. Systemic administration results indelivery of a pharmaceutical composition to essentially the entireperipheral nervous system of the subject and may also result in deliveryto the central nervous system depending on the properties of thecomposition.

Routes of peripheral administration useful in the methods of theinvention encompass, without limitation, oral administration, topicaladministration, intravenous or other injection, and implanted minipumpsor other extended release devices or formulations. A pharmaceuticalcomposition useful in the invention can be peripherally administered,for example, orally in any acceptable form such as in a tablet, liquid,capsule, powder, or the like; by intravenous, intraperitoneal,intramuscular, subcutaneous or parenteral injection; by transdermaldiffusion or electrophoresis; topically in any acceptable form such asin drops, creams, gels or ointments; and by minipump or other implantedextended release device or formulation.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES Example 1 High-throughput Screening Assay for Identification ofInhibitors of Persistent Sodium Current

To identify compounds that inhibit persistent sodium current, a primaryhigh-throughput screen was employed, see, e.g., Joseph S. Adorante etal., High-throughput Screen for Identifying Channel Blockers thatSelectively Distinguish Transient from Persistent Sodium Channels, U.S.Patent Publication No. 2002/0077297 (Jun. 20, 2002), which is herebyincorporated by reference in its entirety.

I. Compound Identification Assay Overview

To examine the ability of test compounds to alter persistent sodiumcurrent, human embryonic kidney (HEK) cells were transfected withNa_(v)1.3 sodium channel to obtain cells that express sodium currentcapable of mediating persistent sodium current. HEK cells expressingNa_(v)1.3 (HEK-Na_(v)1.3) were added to assay plate wells containing aNa⁺-free media and physiologic concentrations of K+ (4.5 mM) andpreincubated for 20 minutes with ion-sensitive FRET dyes and either 5 μMof a test compound or a DMSO control. The assay plates were thentransferred to a voltage/ion probe reader (VIPR) (Aurora Biosciences,San Diego, Calif.) and the VIPR was adjusted so that the fluorescentemission ratio from the donor ands acceptor FRET dyes equaled 1.0. Toelicit persistent sodium current, a double addition protocol wasperformed by first adding an isotonic solution to adjust theconcentration of sodium and potassium ions in the well to 110 mM and 10mM, respectively, and measuring the resulting sodium-dependentdepolarization and second by adding K⁺ to a final concentration of 80mM, and measuring potassium-dependent depolarization. Test compoundsthat block the Na⁺ dependent signal, but not the K⁺ dependent signalwere selected for further analysis. The Na⁺-dependent depolarizationresulting from the persistent Na⁺ was measured as shown in FIG. 2. Thelabeled boxes indicate the application of Na⁺ or K⁺. Circles indicatethe control response with 0.1% DMSO added, triangles show the effects ofthe Na⁺ channel inhibitor tetracaine (10 μM), and the diamonds show theresponse during the application of a non-specific channel blocker.

In this high-throughput assay, non-specific agents that inhibit membranedepolarizations induced by any effector must be distinguished from truepersistent Na⁺ current antagonists, which block only Na⁺-dependentdepolarizations. Therefore, a counter-screen to determine the ability ofcompounds to alter K⁺-dependent depolarization was performed. As shownin FIG. 2, following pre-incubation with vehicle alone (DMSO) both Na⁺and K⁺ additions produced a robust depolarization as indicated by theincrease in Rf/Ri. Tetracaine, a Na⁺ channel blocker, inhibited theNa⁺-dependent, but not the K⁺-dependent change in Rf/Ri. In contrast, anon-specific inhibitor of Na⁺ and K⁺-dependent depolarization blockedthe change in Rf/Ri following either addition. This data demonstratesthat selective antagonists of the persistent sodium current can beidentified using the described method.

To eliminate compounds that non-specifically inhibited the Na⁺-dependentdepolarization, data obtained using the above procedure was analyzedwith respect to a counter-screen that used K⁺-dependent depolarizationas a readout. To select hits from the primary screen, the data wereplotted as histograms. Inhibition of the Na⁺-dependent depolarizationwas plotted against inhibition of the K⁺-dependent depolarization. Basedon these data, the criteria for selection as a hit, was a greater orequal to 90% inhibition of the Na⁺-dependent depolarization and a lessthan or equal to 20% inhibition of the K⁺-dependent depolarization. Thisprotocol provided a distinction between compounds that were inert ornon-specific in their effects and compounds that specifically block thepersistent sodium current.

II. Solutions

Solution compositions and volumes used in the assay are described below.Functions of some components of the solutions using the assay are asfollows: (1) CC2-DMPE: a stationary coumarin-tagged phospholipidresonance energy donor. This dye is excited at 405 nm wavelength lightand in the absence FRET emits fluorescence at 460 nm. (2) DiSBAC2 (3) orDiSBAC6(3): mobile resonance energy acceptors that partition across themembrane as a function of the electric field. The excitation spectra forthese dyes overlap the emission of the coumarin donor and, thus, theyact as FRET acceptors. They have an emission spectrum in the range of570 nm. (3) ESS-AY17: reduces the background fluorescence thatcomplicates the assay. (4) CdCl₂ (400 μM) was included in thepre-incubation solutions to stabilize the membrane potential of thecells at negative resting potential, resulting in the maximum number ofNa⁺ channels being available for activation. (5) Extracellular Cl— wasreplaced with MeSO₃ during preincubation and throughout the assay. Thiseliminates a complicating Cl— current during the assay and results in anamplified and more stable voltage-change induced by the persistent Na⁺current. (6) 1st K⁺ addition: functions to depolarize the test cells toa voltage that activates substantial numbers of Na⁺ channels. (7) 2nd K⁺addition: this addition produces a K⁺-dependent depolarization, which isused as a counterscreen to eliminated non-specific blockers.

III. Cell Culture

HEK-293 cells were grown in Minimum Essential Medium (Invitrogen, Inc.,Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen,Inc., Carlsbad, Calif.) and 1% Pennicillin-Streptomycin (Invitrogen,Inc., Carlsbad, Calif.). Medium for HEK-Na _(v)1.3 cells also contained500 mg/ml G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μMTTX (Calbiochem, Inc., San Diego, Calif.) for maintaining selectivepressure. Cells were grown in vented cap flasks, in 90% humidity and 10%CO2, to about 80% confluence and generally split by trypsinization 1:5or 1:10.

HEK-Na_(v)1.3 cells were seeded in 96-well plates (Becton-Dickinson, SanDiego, Calif.) coated with Matrigel (Becton-Dickinson, San Diego,Calif.) at 40,000 cells (in 100 μl culture medium) per well, and assayedthe following day (16-20 hours). Cells were sometimes incubated in96-well plates at somewhat lower densities (20,000 per well), andincubated for up to 40-48 hours.

IV. HEK-Na_(v)1.3 Handling and Dye Loading

Approximately 16 to 24 hours before the assay, HEK-Na_(v)1.3 cells wereseeded in 96-well poly-lysine coated plates at 40,000 per well. On theday of the assay, medium was aspirated and cells were washed 3 timeswith 150 uL of Bath Solution #1 (BS#1) using CellWash (ThermoLabSystems, Franklin, Mass.).

A 20 μM CC2-DMPE solution was prepared by mixing coumarin stock solutionwith 10% Pluronic 127 1:1 and then dissolving the mix in the appropriatevolume of BS#1. After the last wash, 50 ml of 20 μM CC2-DMPE solutionwas added to 50 mL of residual bath in each well to make 10 μM coumarinstaining buffer. Plates were incubated in the dark for 30-60 minutes atroom temperature.

While the cells were being stained with coumarin, a 10 μM DiSBAC2(3)solution in TEA-MeSO3 bath was prepared. In addition to oxonol, thissolution contained any drug(s) being tested, at 4 times the desiredfinal concentration (e.g. 20 μM for 5 μM final), 1.0 mM ESS-AY17, and400 μM CdCl₂.

After 30-60 minutes of CC2-DMPE staining, the cells were washed 3 timeswith 150 μL of TEA-MeSO₃ buffer. Upon removing the bath, the cells wereloaded with 80 μL of the DiSBAC2(3) solution and incubated for 20-30minutes as before. Typically, wells in one column on each plate (e.g.column 11) were free of test drug(s) and served as positive and negativecontrols.

Once the incubation was complete, the cells were ready to be assayed onVIPR for sodium addback. 240 μL of NaMeSO3 buffer was added to stimulatethe cells, resulting in a 1:4 dilution of the drugs; 240 μL of TEA-MeSO₃buffer or 1 μM TTX was used as a positive control.

V. VIPR Instrumentation and Data Process

Optical experiments in microtiter plates were performed on theVoltage/ion Probe Reader (VIPR) using two 400 nm excitation filters andfilter sticks with 460 nm and 570 nm filters on the emission side forthe blue and red sensitive PMTs, respectively. The instrument was run incolumn acquisition mode with 2 or 5 Hz sampling and 30 seconds ofrecording per column. Starting volumes in each well were 80 ml; usually240 mL was added to each well during the course of the experiment. Thelamp was allowed to warm up for about 20 minutes, and power to the PMTswas turned on for about 10 minutes prior to each experiment.

Ratiometric measurements of changes in fluorescent emissions at 460- and570 nm on the VIPR platform (Aurora Bioscience, San Diego, Calif.)demonstrated that this assay format produces a robust and reproduciblefluorescent signal upon depolarization of HEK-Na_(v)1.3 cells with aNa⁺/K⁺ addition. From a normalized ratio of 1.0 in Na⁺-free media,Na⁺-dependent depolarization resulted in an increase in the 460/570ratio to over 2.2 (FIG. 2). Inter-well analysis of the ratios indicatedthat the amplitude of signal was large enough and consistent enough tobe used in high-throughput screening.

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The VIPR sampling ratevaried between 2 and 5 Hz in different experiments, with 5 Hz used forhigher resolution of the peak sodium responses. The process ofcalculating these ratios was performed as follows. On all plates, column12 contained TEA-MeSO₃ buffer with the same DiSBAC2(3) and ESS-AY17concentrations as used in the cell plates; however no cells wereincluded in column 12. Intensity values at each wavelength were averagedfor the duration of the scan. These average values were subtracted fromintensity values in all assay wells. The initial ratio obtained fromsamples 5-10 (Ri) was defined as:

${Ri} = \frac{{Intensity}_{{460\; n\; m},{{samples}\mspace{14mu} 5\text{-}10}} - {background}_{460n\; m}}{{Intensity}_{{580\; n\; m},{{samples}\mspace{14mu} 5\text{-}10}} - {background}_{580\; n\; m}}$

and the ratio obtained from sample f (Rf) was defined as:

${Rf} = \frac{{Intensity}_{{460\; n\; m},{{samples}\mspace{14mu} f}} - {background}_{460n\; m}}{{Intensity}_{{580\; n\; m},{{samples}\mspace{14mu} f}} - {background}_{580\; n\; m}}$

Final data were normalized to the starting ratio of each well andreported as Rf/Ri. The fluorescent response in the Na_(v)1.3 persistentcurrent assay reached a peak approximately 10 seconds following thestart of the run, therefore, the maximum ratio was selected as thereadout for the assay (FIG. 3).

VI. Assay Reproducibility and Resolution

The assay format described above allows for quality assurance bymeasuring both negative (DMSO 0.1%) and positive (tetracaine 10 μM)controls. Every 10th plate in an assay run was a control plate. The datafrom these plates were used to verify that the assay conditions wereoptimal and to normalize the data from the test compounds. FIG. 3 showsresults from control plates from multiple assays.

In FIG. 3, control plates having wells containing either 0.1% DMSO or 10μM tetracaine were run after every ninth assay plate. The response toNa⁺-dependent depolarization was measured and the data were binned intohistograms as shown. The mean maximum response (Max) obtained in thepresence of (0.1% DMSO) and the mean minimum response (Min) obtained inthe presence of 10 μM tetracaine were determined. For quality control,data variance was compared to the difference between the maximum andminimum signals. This was accomplished by calculating a screening window(z) for each control plate. Data for the run was accepted if 1.0≧Z≧0.5.

$Z = {1 - \frac{{3 \times {STD}_{\max}} + {3 \times {STD}_{\min}}}{{Mean}_{\max} - {Mean}_{\min}}}$

Example 2 Moderate-throughput Screening Assay for Selectivity ofInhibitors of Persistent Sodium Current

Compounds obtained by the high-throughput screening described in Example1 were tested for selectivity of blockade of persistent sodium currentwith respect to blockade of transient sodium current using amoderate-throughput screen. The selectivity assay utilizes Estimtechnology (Aurora Bioscience, San Diego, Calif.) to induce channelactivation. This assay has an inherently greater time resolution thanthe high-throughput assay, and thus allows the measurement of both thetransient and persistent components of the Na⁺ currents within a singleexperiment.

I. Compound Selectivity Assay Overview

The Estim technology involves instrumenting 96-well plates withelectrodes so that application of an appropriate voltage gradient acrossthe well (electric field stimulation, EFS) can be used for activation ofthe ion channels in the target cells. EFS of HEK-293 cells expressingNa_(v)1.3 channels resulted in a rapid depolarization followed by adelayed repolarization. The transient Na⁺ current drives the rapiddepolarization while the persistent Na⁺ current sustains the delayedrepolarization. When similar experiments were performed in cellsexpressing channels that do not exhibit persistent currents, only rapiddepolarization was seen. For quantification of the block of transientcurrent, the amplitude of peak response was averaged for seven stimuli.The average response was converted to activity by normalizing againstthe difference between the responses in Ringer's solution with DMSO andRinger's solution containing 10 μM tetracaine. Persistent currentactivity was calculated by integrating under the curve. The areaobtained for each compound was normalized against the responses obtainedwith the DMSO control and in the presence of 10 μM tetracaine.

II. Cell Culture

Approximately 16 to 24 hours before the assay, HEK-Na_(v)1.3 cells wereseeded in 96-well poly-lysine coated plates at 60,000 per well. On theday of the assay, medium was aspirated were cells were washed 3 timeswith 150 μL of HBSS using CellWash (Thermo LabSystems, Franklin, Mass.).

III. HEK-Na_(v)1.3 Handling and Dye Loading

A 20 μM CC2-DMPE solution was prepared by mixing coumarin stock solutionwith 10% Pluronic 127 1:1 and then dissolving the mix in the appropriatevolume of HBSS. After the last wash, 50 μL of 20 μM CC2-DMPE solutionwas added to 50 μL of residual bath in each well to make 10 μM coumarinstaining buffer. Plates were incubated in the dark for 30 minutes atroom temperature.

While the cells were being stained with CC2-DMPE, a 0.2 μM DiSBAC6(3)solution in HBSS was prepared.

After 30 minutes of CC2-DMPE staining, the cells were washed 3 timeswith 150 μL of HBSS. After the last wash, 50 μL of 0.2 μM DiSBAC6(3)solution was added to 50 μL of residual bath in each well to make 0.1 μMoxonol staining buffer. Plates were then incubated in the dark for 15minutes.

After 15 minutes of DiSBAC6(3) staining, the cells were washed again 3times with 150 μL of HBSS. After the last wash, 50 μL of 1.0 μM ESS-AY17solution was added to 50 μL of residual bath in each well to make 0.5 μMESS. This solution also contained any drug(s) being tested, at twice thedesired final concentrations. Plates were incubated in the dark againfor 15 minutes. Once the incubation was complete, the cells were assayedon EFS/VSP reader.

III. Fast FRET Reader Instrumentation and Data Process

Optical experiments in microtiter plates were performed on the fast FRETReader using two 400 nm excitation filters and filter sticks with 460 nmand 580 nm filters on the emission side for the blue and red sensitivePMTs, respectively. The instrument was run in column acquisition modewith 100 Hz sampling and 12 seconds of recording per column. Sevenpulses were applied at 1 Hz, starting at 2 seconds. The lamp was allowedto warm up for about 20 minutes, and power to the PMTs was turned on forabout 10 minutes prior to each experiment.

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The process of calculatingthese ratios was performed as follows. On all plates, column 12contained. HBSS with the same ESS-AY17 concentration as used in the cellplates; however no cells were included in column 12. Intensity values ateach wavelength were averaged for the duration of the scan. Theseaverage values were subtracted from intensity values in all assay wells.The initial ratio obtained from samples 50-100 (Ri) was defined as:

${Ri} = \frac{{Intensity}_{{460\; n\; m},{{samples}\mspace{14mu} 50\text{-}100}} - {background}_{460n\; m}}{{Intensity}_{{580\; n\; m},{{samples}\mspace{14mu} 50\text{-}100}} - {background}_{580\; n\; m}}$

and the ratio obtained from sample f (Rf) was defined as:

${Rf} = \frac{{Intensity}_{{460\; n\; m},{{samples}\mspace{14mu} f}} - {background}_{460n\; m}}{{Intensity}_{{580\; n\; m},{{samples}\mspace{14mu} f}} - {background}_{580\; n\; m}}$

Data were normalized to the starting ratio of each well and reported asRf/Ri. The transient Na⁺-current signal was calculated as average of thepeaks resulting from the seven electric pulses applied in the course ofrecording. The persistent Na⁺-current signal was calculated integratingthe area under the total response during the seven electric pulsesapplied in the course of recording. Selectivity was determined bycomparison of concentrations of agent required to block 50% of thepersistent current (IC₅₀) vs. the IC₅₀ for the transient current.

Example 3 Electrophysiological Assay for Selectivity of Inhibitors ofPersistent Sodium Current

To confirm the blocking selectivity of test compounds for persistentsodium current, individual compounds were examined using a whole-cellpatch clamp method.

HEK cells transfected with Na_(v)1.3 sodium channels that expresstransient and persistent sodium currents were plated onto glasscoverslips and cultured in MEM cell culture media with Earle's salts andGlutaMAX (Invitrogen, Inc., Carlsbad, Calif.) supplemented with: 10%Fetal bovine serum, heat inactivated (Invitrogen, Inc., Carlsbad,Calif.), 0.1 mM MEM non-essential amino acids (Invitrogen, Inc.,Carlsbad, Calif.), 10 mM HEPES (Invitrogen, Inc., Carlsbad, Calif.), 1%Penicillin/Streptomycin (Invitrogen, Inc., Carlsbad, Calif.).

After an incubation period of from 24 to 48 hours the culture medium wasremoved and replaced with external recording solution (see below). Wholecell patch clamp experiments were performed using an EPC10 amplifier(HEKA Instruments, Lambrecht, Germany.) linked to an IBM compatiblepersonal computer equipped with PULSE software. Borosilicate glass patchpipettes were pulled to a fine tip on a P90 pipette puller (SutterInstrument Co., Novato, Calif.) and were polished (Microforge,Narishige, Japan) to a resistance of about 1.5 Mohm when filled withintracellular recording solution (Table 1).

TABLE 1 Patch Clamp Solutions External Recording Solution InternalRecording Solution Compound Concentration Compound Concentration NaCl127 mM CsMeSO₃ 125 mM HEPES (free acid) 10 mM CsCl 25 mM KCl 5 mMNaHEPES 10 mM CsCl 5 mM Amphotericin 240 μg/ml Glucose 10 mM MgCl₂ 0.6mM CaCl₂ 1.2 mM CdCl₂ 200 μM pH to 7.4 with NaOH @ pH 7.20 with CsOH300mOsm room temp. 290 mOsm.

Persistent and transient currents in HEK cells expressing Na_(v)1.3channels were measured by applying 200-msec depolarizations from aholding potential of −90 mV to 0 mV. Background currents that remainedin the presence of 500 nM TTX were subtracted from all traces. Drugswere perfused directly into the vicinity of the cells using amicroperfusion system.

Under control conditions, depolarizing pulses elicited a large transientinward current that declined to a smaller persistent current, whichremained stable during the remainder of the pulse (FIG. 4, control).Addition of 500 nM TTX completely blocked both the transient andpersistent currents (FIG. 4, TTX). Application of 3 μM of Compound 1,produced a much different effect. Inspection of FIG. 4 reveals that theCompound 1 blocked 99% of the persistent current while only reducing thetransient current by 16%. Dose-response analysis for Compound 1demonstrates its significant selectivity for blocking the persistentsodium current relative to the transient sodium current over a fourorder of magnitude range (FIG. 5).

Example 4 Administering a Selective Persistent Sodium Current Antagonistin a Rodent Model Results in Reduced Epileptic Seizures

This study examined the anti-seizure efficacy of Compound 1 against toreference compounds (Diazepam and Sipatrigine) using the audiogenicmouse model as the test platform.

DBA mice are well established in the literature as a model foraudiogenic seizures (AGS). This genetically based model is attractivefor testing potential therapeutics in that no treatment protocol isrequired to create the condition. It is classified as inheritedidiopathic epilepsy with no known associated organic disease.

The AGS response in these mice consists of a progressive sequence ofbehaviors. The latency to onset may vary from 2 to 15 seconds, and isage variant. The initial wild running phase may be divided into an earlyrunning phase that varies from 5 tp 20 seconds, followed by a wildrunning phase that continues for 10-20 seconds. Wild running mayprogress to tonic/clonic seizures, with loss of righting ability,respiratory suppression and death.

We a scoring system to quantify the effects of the test compounds on theinduction of seizures. The behavioral sequence was assigned ascendingnumerical values (Table 1), and the highest numerical value reached byan animal was that animal's score. Results are reported as an averagescore for ten animals in a treatment group.

TABLE 1 Seizure Scale 1 Staring 2 Head/body tremors/jerks 3 Toniccontraction/Straub tail 4 Wild running 5 Wild running/Jumping 6 TonicClonic Seizure 7 Convulsion 8 Death

Animals. Male DBA/2 mice were obtained from Jackson Laboratories at 21day post partum. The animals were acclimated for 1 or 2 days prior touse. Individual animal weights were recorded immediately beforetreatment. Surviving animals were euthanized within three days ofcompletion of testing.

Seizure Induction. Mice were placed in a test chamber (25 cm i.d.) andexposed to pure tone sound of 11 kHz at a minimum of 116 dB forapproximately 60 s until a sequential seizure response, consisting of anearly wild running phase, followed by generalized myoclonus and tonicflexion and extension, was obtained. Non-responders were challenged upto five times. Ten animals were obtained by this method for eachintended treatment group, with a standard deviation in their maximumscore of less than 25%. The acoustic stimulus signal was produced usinga signal generator and projected via four high-frequency ceramicspeakers mounted on the roof of the chamber. Chamber calibration wasperformed daily to ensure consistent sound pressure. AGS behaviors werevideotaped for later analysis.

I.P. injections: The 10 animals in each treatment group were injectedintra-peritoneally with the test drug in a volume of 10 mL/kg of bodyweight. Injections were 60 min prior to sound challenge.

Data analyses: Data were tested for normal distribution, and (whennormal) were analyzed using ANOVA followed by Student's t test. When thedata were not normally distributed, analysis was with the Mann-Whitneyrank sum test. Significance was set at p<0.05.

Results: Mean AGS scores for each group (with standard deviations) areshown for each treatment in Table 2. No control animals experiencedfull-blown convulsive seizures. Control values near five indicate thatthese animals entered the wild running phase and either did not progressto convulsions or exhibited infrequent or mild tonic-clonic seizures.Control animals exhibited tonic body contraction (back arching, hind legrigidity), running and some body clonus (jerks, tremors). The responsewas quite variable with approximately 20% of the animals exhibiting verymild (if any) symptoms. No control animals developed powerful TCSsymptoms that progressed to death.

Although the lack of a full-blown seizure response in the controlanimals limited the power of this study, compounds (Diazepam andSipatrigine) that are known to reduce the behavioral response tosound-induced seizures in DBA mice were effective at the predicteddoses. Compound 1 (1 mg/kg) also significantly reduced seizure responseat 60 minutes after dosing. The estimated plasma concentration forCompound 1 at this time would be on approximately 5 μM, a concentrationthat should reduce the persistent sodium current by more than 60% whilehaving only a limited effect on the transient current. The fact thatstatistically significant effects on seizure response were detected atthis concentration in spite of both the high variability and the reducedtherapeutic window in this assay indicates the efficacy of Compound 1derives from its effect on the persistent sodium current. Table 2.Reduction of seizure score in DBA mice following treatment withpotential anti-epileptic compounds.

TABLE 2 Reduction of seizure score in DBA mice following treatment withpotential anti-epileptic compounds. Dose Treatment (mg/kg) Mean ± s.d. pControl — 4.8 ± 1.9 Diazepam 0.15 3.1 ± 1.1 0.02 Sipatrigine 3 3.3 ± 1.70.04 Compound 1 1 3.2 ± 2.3 0.04

Example 5 Synthesis of Exemplary Compounds Representative of Formula 1

A compound having general Formula 1, exemplified bythiophene-2-carboxylic acid (4-phenyl-butyl)-amide (Compound 1; FIG. 1)can be prepared as follows. A solution of thiophene-2-carbonyl chloride(147 mg, 1.0 mmol), triethylamine (101 mg, 1.0 mmol) in dichloromethaneis treated with 4-phenylbutylamine (149 mg, 1.0 mmol). The reactionmixture is stirred until no further reaction occurs and is quenched bythe addition of aqueous NaHCO₃ solution. The organic phase is collectedand concentrated to give the title compound.

Example 6 Synthesis of Exemplary Compounds Representative of Formula 2

A compound having general Formula 2, exemplified by1-Benzyl-4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-pyridine (Compound 2;FIG. 1) can be prepared as follows. A solution of4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-pyridine (223 mg, 1.0 mmol) isprepared by the method of H. Smith Broadbent, et al., Quinoxalines. I.Preparation and Stereochemistry of Decahydroquinoxalines, 82(1) J. AMER.CHEM. SOC. 189-193 (1960) in chloroform is treated with benzylbromide(171 mg, 1.0 mmol). The reaction is stirred until no further reactionoccurs. The reaction mixture is concentrated to give the title compound.

Example 7 Synthesis of Exemplary Compounds Representative of Formula 3

A compound having general Formula 3, exemplified by6-Isopropyl-3-methyl-2-{4-[(4-propoxy-benzylidene)-amino]-benzylidene}-cyclohexanone(Compound 3; FIG. 1) can be prepared as follows. A solution of menthone(154 mg, 1.0 mmol) and 4-aminobenzaldehyde (121 mg, 1.0 mmol) indimethylsulfoxide is treated with potassium hydroxide (56 mg, 1.0 mmol).The reaction is stirred until no futher reaction occurs. The reactionmixture is poured into ethyl acetate and water. The organic phase iscollected, dried and concentrated to give2-(4-Amino-benzylidene)-6-isopropyl-3-methyl-cyclohexanone. The2-(4-Amino-benzylidene)-6-isopropyl-3-methyl-cyclohexanone is dissolvedin dichloromethane and treated with 4-propoxybenzaldehyde (164 mg, 1.0mmol) and anhydrous Na₂SO₄. The reaction mixture is stirred until nofurther reaction occurs. The reaction mixture is filtered andconcentrated to give the title compound.

Example 8 Synthesis of Exemplary Compounds Representative of Formula 4

A compound having general Formula 4, exemplified by3-(2,2,2-Trifluoro-acetylamino)-benzoic acid 2-oxo-2-phenyl-ethyl ester(Compound 4; FIG. 1) can be prepared as follows. A solution of3-aminobenzoic acid (137 mg, 1.0 mmol) in dichloromethane is treatedwith trifluoroacetic anhydride (420 mg, 2.0 mmol). The reaction mixtureis stirred until no further reaction occurs. The reaction mixture isconcentrated to give 3-(2,2,2-Trifluoro-acetylamino)-benzoic acid. Asolution of 3-(2,2,2-Trifluoro-acetylamino)-benzoic acid (233 mg, 1.0mmol) and 2-hydroxyacetophenone (136 mg, 1.0 mmol) in dimethylformamideand diisopropylethylamine (260 mg, 2.0 mmol) is treated with HBTU (379mg, 1.0 mmol). The reaction mixture is stirred until no further reactionoccurs. The reaction is poured into ethyl acetate and water. The organicphase is collected, dried and concentrated to give the title compound.

Example 9 Oral Administration of a Persistent Sodium Current Blocker toTreat Epilepsy

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat a neuropathy. While the example illustrates theuse of a PSCB to treat eplilepsy, any neuropathic condition resultingfrom aberrant activity of a persistent current, such as, e.g., headache,pain, inflammatory diseases, movement disorders, tumors, birth injuries,developmental abnormalities, neurocutaneous disorders, autonomicdisorders, and paroxysmal disorders, can also be treated using thismethod.

A patient presents neuropathic symptoms that are diagnosed as anepilepsy. The patient is treated orally with a therapeutically-effectiveamount of a pharmaceutically acceptable composition comprising a PSCBover a period of several months. The patent is reassessed after thistreatment and it is found that the patient's epileptic seizures havesubside. Repeated administration of the PSCB composition maintains thissustained relief from epileptic seizures.

Example 10 Oral Administration of a Persistent Sodium Current Blocker toTreat Cerebral Hypoxia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat a hypoxia. While the example illustrates the useof a PSCB to treat cerebral hypoxia, any hypoxia resulting from a lossof oxygen to a portion of the body, such as, e.g., diffusion hypoxia,hypoxic hypoxia, cell hypoxia, ischemic hypoxia, or any other accidentalor purposeful reduction or elimination of oxygen supply to a tissue, canalso be treated using this method.

A patient presents symptoms that are diagnosed as cerebral hypoxia. Thepatient is treated orally with a therapeutically-effective amount of apharmaceutically acceptable composition comprising a PSCB over a periodof several months. The patent is reassessed after this treatment and itis found that the patient's symptoms have subside. Continuedadministration of the PSCB composition maintains alleviation of thesesymptoms.

Example 11 Oral Administration of a Persistent Sodium Current Blocker toTreat Cardiac Ischemia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat an ischemia. While the example illustrates theuse of a PSCB to treat myocardiac ischemia, any ischemia resulting froma loss of blood to a portion of the body, such as, e.g., cerebralischemia, myoischemia, diabetes ischemia, ischemia retinae, posturalischemia, or any other accidental or purposeful reduction or completeobstruction of blood supply to a tissue, can also be treated using thismethod.

A patient presents symptoms that are diagnosed as myocardiac ischemia.The patient is treated orally with a therapeutically-effective amount ofa pharmaceutically acceptable composition comprising a PSCB over aperiod of several months. The patent is reassessed after this treatmentand it is found that the patient's symptoms have subside. Continuedadministration of the PSCB composition maintains alleviation of thesesymptoms.

Example 12 Oral Administration of a Persistent Sodium Current Blocker toTreat Multiple Sclerosis

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat multiple sclerosis.

A patient presents symptoms that are diagnosed as multiple sclerosis.The patient is treated orally with a therapeutically-effective amount ofa pharmaceutically acceptable composition comprising a PSCB over aperiod of several months. The patent is reassessed after this treatmentand it is found that the patient's symptoms have stablize. Continuedadministration of the PSCB composition maintains prevents continuedprogression of the disease.

Example 13 Oral Administration of a Persistent Sodium Current Blocker toTreat Amyotrophic Lateral Sclerosis

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat amyotrophic lateral sclerosis.

A patient presents symptoms that are diagnosed as amyotrophic lateralsclerosis. The patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB over a period of several months. Thepatent is reassessed after this treatment and it is found that thepatient's symptoms have stablize. Continued administration of the PSCBcomposition maintains prevents continued progression of the disease.

Example 14 Oral Administration of a Persistent Sodium Current Blocker toTreat Aberrant Nitric Oxide Levels

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat aberrant nitric oxide levels.

A patient presents symptoms that are diagnosed as aberrant nitric oxidelevels. The patient is treated orally with a therapeutically-effectiveamount of a pharmaceutically acceptable composition comprising a PSCBover a period of several months. The patent is reassessed after thistreatment and it is found that the patient's symptoms have subside.Continued administration of the PSCB composition maintains alleviationof these symptoms.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. Various modifications can be made without departing from thespirit of the invention.

1. A method of treating an epileptic condition in a mammal, comprisingadministering to said mammal an effective amount of a selectivepersistent sodium channel antagonist, wherein said antagonist has atleast 20-fold selectivity for a persistent sodium current relative to atransient sodium current, and wherein said antagonist is a compoundincluded in formula 4, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or racemic mixture thereof:

wherein, Ar³ is phenyl or a substituted phenyl; Ar⁴ is phenyl or asubstituted phenyl; X¹ is absent or selected from the group consistingof

Y¹ is absent or selected from the group consisting of

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, COCF₃ and a C₁ to C₈ alkyl; R¹⁰ and R¹¹ are independentlyselected from the group consisting of hydrogen, hydroxy, halogen, and aC₁ to C₈ alkyl.
 2. The method of claim 1, wherein said persistent sodiumcurrent is Na_(v)1.1 persistent current.
 3. The method of claim 1,wherein said persistent sodium current is Na_(v)1.2 persistent current.4. The method of claim 1, wherein said persistent sodium current isNa_(v)1.3 persistent current.
 5. The method of claim 1, wherein saidpersistent sodium current is Na_(v)1.5 persistent current.
 6. The methodof claim 1, wherein said persistent sodium current is Na_(v)1.6persistent current.
 7. The method of claim 1, wherein said persistentsodium current is Na_(v)1.7 persistent current.
 8. The method of claim1, wherein said persistent sodium current is Na_(v)1.8 persistentcurrent.
 9. The method of claim 1, wherein said persistent sodiumcurrent is Na_(v)1.9 persistent current.
 10. The method of claim 1,wherein said mammal is a human.
 11. The method of claim 1, wherein saidantagonist has at least 50-fold selectivity for said persistent sodiumcurrent relative to said transient sodium current.
 12. The method ofclaim 1, wherein said antagonist has at least 200-fold selectivity forsaid persistent sodium current relative to said transient sodiumcurrent.
 13. The method of claim 1, wherein said antagonist has at least400-fold selectivity for said persistent sodium current relative to saidtransient sodium current.
 14. The method of claim 1, wherein saidantagonist has at least 600-fold selectivity for said persistent sodiumcurrent relative to said transient sodium current.
 15. The method ofclaim 1, wherein said antagonist has at least 1000-fold selectivity forsaid persistent sodium current relative to said transient sodiumcurrent.
 16. The method of claim 1, wherein said antagonist isadministered peripherally.
 17. The method of claim 1, wherein saidantagonist is administered systemically.
 18. The method of claim 1,wherein said antagonist is administered orally.
 19. The method of claim1, wherein said antagonist is administered in a sustained releaseformula.
 20. The method of claim 1, wherein said antagonist isadministered in a bioerodible delivery system.
 21. The method of claim1, wherein said antagonist is administered in a non-bioerodible deliverysystem.
 22. The method of claim 1, wherein said Ar³ is phenyl.
 23. Themethod of claim 1, wherein said Ar³ is a substituted phenyl.
 24. Themethod of claim 23, wherein said substituted phenyl is substituted withone or more of halogen, a C₁ to C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, or(CR⁵R⁶)_(c)N(R⁷)₂ wherein, R⁵ and R⁶ are independently selected from thegroup consisting of hydrogen, hydroxy, fluoro, and a C₁ to C₈ alkyl; R⁷is selected from the group consisting of hydrogen, and a C₁ to C₈ alkyl;and c is 0, 1, 2, 3, 4, or
 5. 25. The method of claim 1, wherein saidAr⁴ is phenyl.
 26. The method of claim 1, wherein said Ar⁴ is asubstituted phenyl.
 27. The method of claim 26, wherein said substitutedphenyl is substituted with one or more of halogen, a C₁ to C₈ alkyl,NO₂, CF₃, OCF₃, OCF₂H, CN, or (CR⁵R⁶)_(c)N(R⁷)₂ wherein, R⁵ and R⁶ areindependently selected from the group consisting of hydrogen, hydroxy,fluoro, and a C₁ to C₈ alkyl; R⁷ is selected from the group consistingof hydrogen, and a C₁ to C₈ alkyl; and c is 0, 1, 2, 3, 4, or
 5. 28. Themethod of claim 1, wherein said R⁸ is hydrogen, methyl, ethyl, propyl,or isopropyl.
 29. The method of claim 1, wherein said R⁹ is hydrogen,methyl, ethyl, propyl, or isopropyl.
 30. The method of claim 1, whereinsaid R¹⁰ is hydrogen, methyl, ethyl, propyl, or isopropyl.
 31. Themethod of claim 1, wherein said R¹¹ is hydrogen, methyl, ethyl, propyl,or isopropyl.
 32. The method of claim 22, wherein said antagonist is3-(2,2,2-Trifluoro-acetylamino) -benzoic acid 2-oxo-2-phenyl-ethylester.
 33. The method of claim 25, wherein said antagonist is


34. The method of claim 1, wherein said epileptic condition is apartial-onset seizure.
 35. The method of claim 34, wherein saidpartial-onset seizure is selected from the group consisting of a simplepartial seizure, a complex partial seizure and a secondarily generalizedtonic-clonic seizure.
 36. The method of claim 1, wherein said epilepticcondition is a generalized-onset seizure.
 37. The method of claim 36,wherein said generalized-onset seizure is selected from the groupconsisting of an absence seizure, a tonic seizure, a clonic seizure, amyoclonic seizure, a primary generalized tonic-clonic seizure, and anatonic seizure.
 38. The method of claim 1, wherein said epilepticcondition is an unclassified seizure.
 39. The method of claim 1, whereinsaid epileptic condition is a localization-related syndrome.
 40. Themethod of claim 1, wherein said epileptic condition is ageneralized-onset syndrome.
 41. The method of claim 1, wherein saidepileptic condition is an inherited epileptic condition.
 42. The methodof claim 41, wherein said inherited epileptic condition is selected fromthe group consisting of an idiopathic epilepsy, a Severe MyoclonicEpilepsy in Infancy (SMEI), a Borderline SMEI (SMEB), and a GeneralizedEpilepsy with Febrile Seizures Plus (GEFS+).
 43. The method of claim 1,wherein said effective amount reduces the symptoms of an epilepticcondition by at least 30%.
 44. The method of claim 1, wherein saideffective amount reduces the symptoms of an epileptic condition by atleast 50%.
 45. The method of claim 1, wherein said effective amountreduces the symptoms of an epileptic condition by at least 70%.
 46. Themethod of claim 1, wherein said effective amount reduces the symptoms ofan epileptic condition by at least 90%.